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b/assets/nf-core-nanoseq_social_preview.svg new file mode 100644 index 00000000..f4059875 --- /dev/null +++ b/assets/nf-core-nanoseq_social_preview.svg @@ -0,0 +1,448 @@ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + image/svg+xml + + + + + + + Nanopore demultiplexing, QC and alignment pipeline + nanoseq + + + + + + + + + + + + + + + + + + + + + + + + + From 891d7e8bd9a72f2d299796c1fa61d6de7719dc83 Mon Sep 17 00:00:00 2001 From: drpatelh Date: Wed, 12 Feb 2020 16:15:28 +0000 Subject: [PATCH 02/22] Update docs --- docs/usage.md | 28 ++++++++++++++++++++-------- 1 file changed, 20 insertions(+), 8 deletions(-) diff --git a/docs/usage.md b/docs/usage.md index ed48630b..1008f3dd 100644 --- a/docs/usage.md +++ b/docs/usage.md @@ -138,12 +138,12 @@ If `-profile` is not specified, the pipeline will run locally and expect all sof ### `--input` -You will need to create a file with information about the samples in your experiment/run before executing the pipeline. Use this parameter to specify its location. It has to be a comma-separated file with 4 columns, and a header row. As shown in the examples below, the accepted format of the file is slightly different if you would like to run the pipeline with or without demultiplexing. +You will need to create a file with information about the samples in your experiment/run before executing the pipeline. Use this parameter to specify its location. It has to be a comma-separated file with 5 columns, and a header row. As shown in the examples below, the accepted format of the file is slightly different if you would like to run the pipeline with or without demultiplexing. #### With basecalling and demultiplexing ```bash -sample,fastq,barcode,genome +sample,fastq,barcode,genome,transcriptome Sample1,,1,mm10 Sample2,,2,mm10 Sample3,,3,hg19 @@ -176,12 +176,24 @@ Sample4,SAM101A4.fastq.gz,,/path/to/local/reference/genome.fa > You will have to specify the `--skip_basecalling` parameter if you wish to bypass the basecalling and demultiplexing steps. -| Column | Description | -|----------|----------------------------------------------------------------------------------------------------------------------------| -| `sample` | Sample name without spaces. | -| `fastq` | Full path to FastQ file if previously demultiplexed. File has to be zipped and have the extension ".fastq.gz" or ".fq.gz". | -| `barcode`| Barcode identifier attributed to that sample when multiplexing samples in integer format. | -| `genome` | Genome fasta for alignment. This can either be a local path, or the appropriate key for a genome available on [AWS-iGenomes](https://ewels.github.io/AWS-iGenomes/) (see [iGenomes config file](../conf/igenomes.config)). If unspecified then the alignment step will be skipped for that sample. | +| Column | Description | +|-----------------|----------------------------------------------------------------------------------------------------------------------------| +| `sample` | Sample name without spaces. | +| `fastq` | Full path to FastQ file if previously demultiplexed. File has to be zipped and have the extension ".fastq.gz" or ".fq.gz". | +| `barcode` | Barcode identifier attributed to that sample when multiplexing samples in integer format. | +| `genome` | Genome fasta file for alignment. This can either be blank, a local path, or the appropriate key for a genome available on [AWS-iGenomes](https://ewels.github.io/AWS-iGenomes/) (see [iGenomes config file](../conf/igenomes.config)). Must have the extension ".fasta" or ".fasta.gz", ".fa" or ".fa.gz". | +| `transcriptome` | Transcriptome fasta/gtf file for alignment. This can either be blank or a local path. Must have the extension .fasta" or ".fasta.gz", ".fa", ".fa.gz", ".gtf", ".gtf.gz". | + +#### Providing a reference genome/transcriptome + +Each sample in your `--input` sample sheet can be mapped to its own reference genome or transcriptome. Please see below for additional details to see how to fill in the `genome` and `transcriptome` columns appropriately: + +* If both `genome` and `transcriptome` are not specified then the mapping will be skipped for that sample. +* If both `genome` and `transcriptome` are specified as local fasta files then the transcriptome will be preferentially used for mapping. +* If `genome` is specified as a local fasta file and `transcriptome` is left blank then mapping will be performed relative to the genome. +* If `genome` is specified as an AWS iGenomes key then the `transcriptome` column can be blank. The associated gtf file for the `transcriptome` will be automatically obtained in order to create a transcriptome fasta file. However, the reads will only be mapped to the transcriptome if `--protocol cDNA` or `--protocol directRNA`. If `--protocol DNA` then the reads will still be mapped to the genome essentially ignoring the gtf file. +* If `genome` is specified as a local fasta file and `transcriptome` is a specified as a local gtf file then both of these will be used to create a transcriptome fasta file. However, the reads will only be mapped to the transcriptome if `--protocol cDNA` or `--protocol directRNA`. If `--protocol DNA` then the reads will still be mapped to the genome essentially ignoring the gtf file. +* If `genome` isnt specified and `transcriptome` is then mapping will be performed relative to the transcriptome only if it is provided as a fasta file. ### `--protocol` From 5607713de323a27a6d7cb93ef2fe031df1ceaa4c Mon Sep 17 00:00:00 2001 From: drpatelh Date: Wed, 12 Feb 2020 16:15:40 +0000 Subject: [PATCH 03/22] Change description --- main.nf | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/main.nf b/main.nf index 07a82cdf..eda5290c 100644 --- a/main.nf +++ b/main.nf @@ -28,7 +28,7 @@ def helpMessage() { Mandatory arguments --input [file] Comma-separated file containing information about the samples in the experiment (see docs/usage.md) - --protocol [str] Specifies the type of data that was sequenced i.e. "DNA", "cDNA" or "directRNA" + --protocol [str] Specifies the type of sequencing i.e. "DNA", "cDNA" or "directRNA" -profile [str] Configuration profile to use. Can use multiple (comma separated) Available: docker, singularity, awsbatch, test and more. From c55247f516331e33c785f912f1845e2405b51981 Mon Sep 17 00:00:00 2001 From: drpatelh Date: Wed, 12 Feb 2020 16:42:50 +0000 Subject: [PATCH 04/22] Update docs --- docs/usage.md | 105 +++++++++++++++++++++++++++++++++++--------------- 1 file changed, 74 insertions(+), 31 deletions(-) diff --git a/docs/usage.md b/docs/usage.md index 1008f3dd..3311d983 100644 --- a/docs/usage.md +++ b/docs/usage.md @@ -138,32 +138,85 @@ If `-profile` is not specified, the pipeline will run locally and expect all sof ### `--input` -You will need to create a file with information about the samples in your experiment/run before executing the pipeline. Use this parameter to specify its location. It has to be a comma-separated file with 5 columns, and a header row. As shown in the examples below, the accepted format of the file is slightly different if you would like to run the pipeline with or without demultiplexing. +You will need to create a file with information about the samples in your experiment/run before executing the pipeline. Use this parameter to specify its location. It has to be a comma-separated file with 5 columns and a header row: -#### With basecalling and demultiplexing +| Column | Description | +|-----------------|----------------------------------------------------------------------------------------------------------------------------| +| `sample` | Sample name without spaces. | +| `fastq` | Full path to FastQ file if previously demultiplexed. File has to be zipped and have the extension ".fastq.gz" or ".fq.gz". | +| `barcode` | Barcode identifier attributed to that sample during multiplexing. Has to be an integer. | +| `genome` | Genome fasta file for alignment. This can either be blank, a local path, or the appropriate key for a genome available in [iGenomes config file](../conf/igenomes.config). Must have the extension ".fasta", ".fasta.gz", ".fa" or ".fa.gz". | +| `transcriptome` | Transcriptome fasta/gtf file for alignment. This can either be blank or a local path. Must have the extension ".fasta", ".fasta.gz", ".fa", ".fa.gz", ".gtf" or ".gtf.gz". | + +#### Providing a reference genome/transcriptome + +Each sample in your `--input` sample sheet can be mapped to its own reference genome or transcriptome. Please see below for additional details required to fill in the `genome` and `transcriptome` columns appropriately: + +* If both `genome` and `transcriptome` are not specified then the mapping will be skipped for that sample. +* If both `genome` and `transcriptome` are specified as local fasta files then the transcriptome will be preferentially used for mapping. +* If `genome` is specified as a local fasta file and `transcriptome` is left blank then mapping will be performed relative to the genome. +* If `genome` is specified as an AWS iGenomes key then the `transcriptome` column can be blank. The associated gtf file for the `transcriptome` will be automatically obtained in order to create a transcriptome fasta file. However, the reads will only be mapped to the transcriptome if `--protocol cDNA` or `--protocol directRNA`. If `--protocol DNA` then the reads will still be mapped to the genome essentially ignoring the gtf file. +* If `genome` is specified as a local fasta file and `transcriptome` is a specified as a local gtf file then both of these will be used to create a transcriptome fasta file. However, the reads will only be mapped to the transcriptome if `--protocol cDNA` or `--protocol directRNA`. If `--protocol DNA` then the reads will still be mapped to the genome essentially ignoring the gtf file. +* If `genome` isnt specified and `transcriptome` is provided as a fasta file then mapping will be performed relative to the transcriptome. + +#### Skip basecalling/demultiplexing + +As shown in the examples below, the accepted format of the file is slightly different if you would like to run the pipeline with or without basecalling/demultiplexing. + +##### With basecalling and demultiplexing + +###### Example `samplesheet.csv` ```bash sample,fastq,barcode,genome,transcriptome -Sample1,,1,mm10 -Sample2,,2,mm10 -Sample3,,3,hg19 -Sample4,,4,/path/to/local/reference/genome.fa +Sample1,,1,mm10, +Sample2,,2,hg19, +Sample3,,3,/path/to/local/reference/genome.fa, +Sample4,,4,,/path/to/local/reference/transcriptome.fa +Sample5,,5,/path/to/local/reference/genome.fa,/path/to/local/reference/transcriptome.gtf +Sample6,,6,, +``` + +###### Example command +```bash +nextflow run nf-core/nanoseq \ + --input samplesheet.csv \ + --protocol cDNA \ + --input_path ./fast5/ \ + --flowcell FLO-MIN106 \ + --kit SQK-DCS109 \ + --barcode_kit EXP-NBD103 \ + -profile ``` -> When multiplexed fastq file is provided where demultiplexing is required without basecalling, the sample sheet can also be specified using this format. But you will need to specify the fastq file in `--input_path` instead of just the directory in this case. +##### With basecalling but not demultiplexing + +###### Example `samplesheet.csv` -#### With basecalling but not demultiplexing +> Only a single sample can be specified if you would like to skip demultiplexing ```bash -sample,fastq,barcode,genome -Sample1,,1,mm10 +sample,fastq,barcode,genome,transcriptome +Sample1,,1,/path/to/local/reference/genome.fa, +``` +###### Example command + +```bash +nextflow run nf-core/nanoseq \ + --input samplesheet.csv \ + --protocol cDNA \ + --input_path ./fast5/ \ + --flowcell FLO-MIN106 \ + --kit SQK-DCS108 \ + --skip_demultiplexing \ + -profile ``` -> You will have to specify the `--skip_demultiplexing` parameter if you wish to bypass the demultiplexing step. +##### Without both basecalling and demultiplexing -#### Without both basecalling and demultiplexing +###### Example `samplesheet.csv` ```bash sample,fastq,barcode,genome @@ -174,26 +227,16 @@ Sample4,SAM101A4.fastq.gz,,/path/to/local/reference/genome.fa ``` -> You will have to specify the `--skip_basecalling` parameter if you wish to bypass the basecalling and demultiplexing steps. - -| Column | Description | -|-----------------|----------------------------------------------------------------------------------------------------------------------------| -| `sample` | Sample name without spaces. | -| `fastq` | Full path to FastQ file if previously demultiplexed. File has to be zipped and have the extension ".fastq.gz" or ".fq.gz". | -| `barcode` | Barcode identifier attributed to that sample when multiplexing samples in integer format. | -| `genome` | Genome fasta file for alignment. This can either be blank, a local path, or the appropriate key for a genome available on [AWS-iGenomes](https://ewels.github.io/AWS-iGenomes/) (see [iGenomes config file](../conf/igenomes.config)). Must have the extension ".fasta" or ".fasta.gz", ".fa" or ".fa.gz". | -| `transcriptome` | Transcriptome fasta/gtf file for alignment. This can either be blank or a local path. Must have the extension .fasta" or ".fasta.gz", ".fa", ".fa.gz", ".gtf", ".gtf.gz". | +###### Example command -#### Providing a reference genome/transcriptome - -Each sample in your `--input` sample sheet can be mapped to its own reference genome or transcriptome. Please see below for additional details to see how to fill in the `genome` and `transcriptome` columns appropriately: - -* If both `genome` and `transcriptome` are not specified then the mapping will be skipped for that sample. -* If both `genome` and `transcriptome` are specified as local fasta files then the transcriptome will be preferentially used for mapping. -* If `genome` is specified as a local fasta file and `transcriptome` is left blank then mapping will be performed relative to the genome. -* If `genome` is specified as an AWS iGenomes key then the `transcriptome` column can be blank. The associated gtf file for the `transcriptome` will be automatically obtained in order to create a transcriptome fasta file. However, the reads will only be mapped to the transcriptome if `--protocol cDNA` or `--protocol directRNA`. If `--protocol DNA` then the reads will still be mapped to the genome essentially ignoring the gtf file. -* If `genome` is specified as a local fasta file and `transcriptome` is a specified as a local gtf file then both of these will be used to create a transcriptome fasta file. However, the reads will only be mapped to the transcriptome if `--protocol cDNA` or `--protocol directRNA`. If `--protocol DNA` then the reads will still be mapped to the genome essentially ignoring the gtf file. -* If `genome` isnt specified and `transcriptome` is then mapping will be performed relative to the transcriptome only if it is provided as a fasta file. +```bash +nextflow run nf-core/nanoseq \ + --input samplesheet.csv \ + --protocol cDNA \ + --skip_basecalling \ + --skip_demultiplexing \ + -profile +``` ### `--protocol` From d4c121ff945e0ee59c80d355c2d3885acfd7fc2c Mon Sep 17 00:00:00 2001 From: drpatelh Date: Wed, 12 Feb 2020 16:54:35 +0000 Subject: [PATCH 05/22] Update docs --- docs/usage.md | 55 +++++++++++++++++++++++++++++++++++++-------------- 1 file changed, 40 insertions(+), 15 deletions(-) diff --git a/docs/usage.md b/docs/usage.md index 3311d983..4921a94f 100644 --- a/docs/usage.md +++ b/docs/usage.md @@ -144,20 +144,20 @@ You will need to create a file with information about the samples in your experi |-----------------|----------------------------------------------------------------------------------------------------------------------------| | `sample` | Sample name without spaces. | | `fastq` | Full path to FastQ file if previously demultiplexed. File has to be zipped and have the extension ".fastq.gz" or ".fq.gz". | -| `barcode` | Barcode identifier attributed to that sample during multiplexing. Has to be an integer. | +| `barcode` | Barcode identifier attributed to that sample during multiplexing. Must be an integer. | | `genome` | Genome fasta file for alignment. This can either be blank, a local path, or the appropriate key for a genome available in [iGenomes config file](../conf/igenomes.config). Must have the extension ".fasta", ".fasta.gz", ".fa" or ".fa.gz". | | `transcriptome` | Transcriptome fasta/gtf file for alignment. This can either be blank or a local path. Must have the extension ".fasta", ".fasta.gz", ".fa", ".fa.gz", ".gtf" or ".gtf.gz". | -#### Providing a reference genome/transcriptome +#### Specifying a reference genome/transcriptome -Each sample in your `--input` sample sheet can be mapped to its own reference genome or transcriptome. Please see below for additional details required to fill in the `genome` and `transcriptome` columns appropriately: +Each sample in the sample sheet can be mapped to its own reference genome or transcriptome. Please see below for additional details required to fill in the `genome` and `transcriptome` columns appropriately: * If both `genome` and `transcriptome` are not specified then the mapping will be skipped for that sample. * If both `genome` and `transcriptome` are specified as local fasta files then the transcriptome will be preferentially used for mapping. * If `genome` is specified as a local fasta file and `transcriptome` is left blank then mapping will be performed relative to the genome. +* If `genome` isnt specified and `transcriptome` is provided as a fasta file then mapping will be performed relative to the transcriptome. * If `genome` is specified as an AWS iGenomes key then the `transcriptome` column can be blank. The associated gtf file for the `transcriptome` will be automatically obtained in order to create a transcriptome fasta file. However, the reads will only be mapped to the transcriptome if `--protocol cDNA` or `--protocol directRNA`. If `--protocol DNA` then the reads will still be mapped to the genome essentially ignoring the gtf file. * If `genome` is specified as a local fasta file and `transcriptome` is a specified as a local gtf file then both of these will be used to create a transcriptome fasta file. However, the reads will only be mapped to the transcriptome if `--protocol cDNA` or `--protocol directRNA`. If `--protocol DNA` then the reads will still be mapped to the genome essentially ignoring the gtf file. -* If `genome` isnt specified and `transcriptome` is provided as a fasta file then mapping will be performed relative to the transcriptome. #### Skip basecalling/demultiplexing @@ -171,9 +171,9 @@ As shown in the examples below, the accepted format of the file is slightly diff sample,fastq,barcode,genome,transcriptome Sample1,,1,mm10, Sample2,,2,hg19, -Sample3,,3,/path/to/local/reference/genome.fa, -Sample4,,4,,/path/to/local/reference/transcriptome.fa -Sample5,,5,/path/to/local/reference/genome.fa,/path/to/local/reference/transcriptome.gtf +Sample3,,3,/path/to/local/genome.fa, +Sample4,,4,,/path/to/local/transcriptome.fa +Sample5,,5,/path/to/local/genome.fa,/path/to/local/transcriptome.gtf Sample6,,6,, ``` @@ -194,13 +194,13 @@ nextflow run nf-core/nanoseq \ ###### Example `samplesheet.csv` -> Only a single sample can be specified if you would like to skip demultiplexing - ```bash sample,fastq,barcode,genome,transcriptome -Sample1,,1,/path/to/local/reference/genome.fa, +Sample1,,1,/path/to/local/genome.fa, ``` +> Only a single sample can be specified if you would like to skip demultiplexing + ###### Example command ```bash @@ -214,17 +214,42 @@ nextflow run nf-core/nanoseq \ -profile ``` +##### With demultiplexing but not basecalling + +###### Example `samplesheet.csv` + +```bash +sample,fastq,barcode,genome,transcriptome +Sample1,,1,mm10, +Sample2,,2,hg19, +Sample3,,3,/path/to/local/genome.fa, +Sample4,,4,,/path/to/local/transcriptome.fa +Sample5,,5,/path/to/local/genome.fa,/path/to/local/transcriptome.gtf +Sample6,,6,, +``` + +###### Example command + +```bash +nextflow run nf-core/nanoseq \ + --input samplesheet.csv \ + --protocol DNA \ + --input_path ./undemultiplexed.fastq.gz \ + --barcode_kit 'NBD103/NBD104' \ + --skip_basecalling \ + -profile +``` + ##### Without both basecalling and demultiplexing ###### Example `samplesheet.csv` ```bash -sample,fastq,barcode,genome +sample,fastq,barcode,genome,transcriptome Sample1,SAM101A1.fastq.gz,,mm10 -Sample2,SAM101A2.fastq.gz,,mm10 -Sample3,SAM101A3.fastq.gz,,hg19 -Sample4,SAM101A4.fastq.gz,,/path/to/local/reference/genome.fa - +Sample2,SAM101A2.fastq.gz,,hg19 +Sample3,SAM101A3.fastq.gz,/path/to/local/genome.fa, +Sample4,SAM101A4.fastq.gz,, ``` ###### Example command From 9645da9548b274c264faedf55bf4183718919e34 Mon Sep 17 00:00:00 2001 From: drpatelh Date: Wed, 12 Feb 2020 17:12:20 +0000 Subject: [PATCH 06/22] Update docs --- docs/usage.md | 46 ++++++++++++++++++++++++---------------------- 1 file changed, 24 insertions(+), 22 deletions(-) diff --git a/docs/usage.md b/docs/usage.md index 4921a94f..aab4a249 100644 --- a/docs/usage.md +++ b/docs/usage.md @@ -11,7 +11,7 @@ * [`-profile`](#-profile) * [`--input`](#--input) * [`--protocol`](#--protocol) -* [Basecalling](#basecalling) +* [Basecalling/Demultiplexing](#basecalling-demultiplexing) * [`--input_path`](#--input_path) * [`--flowcell`](#--flowcell) * [`--kit`](#--kit) @@ -267,23 +267,25 @@ nextflow run nf-core/nanoseq \ Specifies the type of data that was sequenced i.e. "DNA", "cDNA" or "directRNA". -## Basecalling - ### `--input_path` -Path to Nanopore run directory e.g. `fastq_pass/`. When `--skip_basecalling` is specified but not `--skip_demultiplexing`, please specify the path to fastq file e.g. `fastq/multiplexed_sample.fastq.gz` +Path to Nanopore run directory (e.g. `fastq_pass/`) or a basecalled fastq file that requires demultiplexing. The latter can only be provided in conjunction with the `--skip_basecalling` parameter. + +## Basecalling/Demultiplexing ### `--flowcell` -Flowcell used to perform the sequencing e.g. `FLO-MIN106`. Not required if `--guppy_config` is specified. +Flowcell used to perform the sequencing e.g. "FLO-MIN106". Not required if `--guppy_config` is specified. ### `--kit` -Kit used to perform the sequencing e.g. `SQK-LSK109`. Not required if `--guppy_config` is specified. +Kit used to perform the sequencing e.g. "SQK-LSK109". Not required if `--guppy_config` is specified. ### `--barcode_kit` -Barcode kit used to perform the sequencing e.g. `SQK-PBK004`. When `--skip_basecalling` is specified but not `--skip_demultiplexing`, please specify the barcoding kit that can be recognised by `qcat`. +Barcode kit used to perform the sequencing e.g. "SQK-PBK004". + +If you would like to skip the basecalling (`--skip_basecalling`) but still perform the demultiplexing please specify a barcode kit that can be recognised by [qcat](https://github.com/nanoporetech/qcat): | `qcat` barcode kit specifications | description | |-----------------------------------|-------------------------------------------------------------------------------| @@ -302,40 +304,40 @@ Barcode kit used to perform the sequencing e.g. `SQK-PBK004`. When `--skip_basec ### `--guppy_config` -Guppy config file used for basecalling passed with the `--config` parameter. Cannot be used in conjunction with `--flowcell` and `--kit`. -This can be a local file (i.e. `/your/dir/guppy_conf.cfg`) or a string specifying a configuration stored in the `/opt/ont/guppy/data` directory of Guppy. +Config file used for basecalling that will be passed to Guppy via the "--config" parameter. Cannot be used in conjunction with `--flowcell` and `--kit`. +This can be a local file (i.e. `/your/dir/guppy_conf.cfg`) or a string specifying a configuration stored in the `/opt/ont/guppy/data/` directory of Guppy. ### `--guppy_model` -Custom basecalling model file (`json`) to pass to Guppy for basecalling with the `--model` parameter. Custom basecalling models can be trained with software such as [Taiyaki](https://github.com/nanoporetech/taiyaki). This can also be a string specifying a model stored in the `/opt/ont/guppy/data` directory of Guppy. +Custom basecalling model file in `json` format that will be passed to Guppy via the "--model" parameter. Custom basecalling models can be trained with software such as [Taiyaki](https://github.com/nanoporetech/taiyaki). This can also be a string specifying a model stored in the `/opt/ont/guppy/data` directory of Guppy. ### `--guppy_gpu` -Whether to demultiplex with Guppy in GPU mode. +Whether to demultiplex with Guppy in GPU mode (default: false). ### `--guppy_gpu_runners` -Number of '--gpu_runners_per_device' used for guppy when using `--guppy_gpu` (default: 6) +Number of "--gpu_runners_per_device" used for Guppy when using `--guppy_gpu` (default: 6). ### `--guppy_cpu_threads` -Number of '--cpu_threads_per_caller' used for guppy when using `--guppy_gpu` (default: 1) +Number of "--cpu_threads_per_caller" used for Guppy when using `--guppy_gpu` (default: 1). ### `--gpu_device` -Basecalling device specified to Guppy in GPU mode using `--device` (default: 'auto') +Basecalling device specified to Guppy in GPU mode using "--device" (default: 'auto'). ### `--gpu_cluster_options` -Cluster options required to use GPU resources (e.g. '--part=gpu --gres=gpu:1') +Cluster options required to use GPU resources (e.g. '--part=gpu --gres=gpu:1'). ### `--qcat_min_score` -Specify the minimum quality score for `qcat` in the range 0-100 (default: 60) +Specify the minimum quality score for qcat in the range 0-100 (default: 60). ### `--qcat_detect_middle` -Search for adapters in the whole read by applying the '--detect-middle' parameter in `qcat` (default: false) +Search for adapters in the whole read by applying the '--detect-middle' parameter in qcat (default: false). ### `--skip_basecalling` @@ -343,13 +345,13 @@ Skip basecalling with Guppy ### `--skip_demultiplexing` -Skip demultiplexing with Guppy or with qcat +Skip demultiplexing with Guppy/qcat ## Alignment ### `--stranded` -Specifies if the data is strand-specific. Automatically activated when using --protocol directRNA (default: false) +Specifies if the data is strand-specific. Automatically activated when using `--protocol directRNA` (default: false). When using `--protocol`/`--stranded` the following command-line arguments will be set for `minimap2` and `graphmap2`: @@ -362,15 +364,15 @@ When using `--protocol`/`--stranded` the following command-line arguments will b ### `--aligner` -Specifies the aligner to use (available are: `graphmap2` or `minimap2`) +Specifies the aligner to use i.e. `graphmap2` or `minimap2`. ### `--save_align_intermeds` -Save the `.sam` files from the alignment step - not done by default +Save the `.sam` files from the alignment step - not done by default. ### `--skip_alignment` -Skip alignment and subsequent process +Skip alignment and downstream processes. ## Coverage tracks From 55b9de984fce186a3ad53f0a2cfdf5573a6ea0f6 Mon Sep 17 00:00:00 2001 From: drpatelh Date: Wed, 12 Feb 2020 17:14:25 +0000 Subject: [PATCH 07/22] Update usage --- docs/usage.md | 6 +++--- 1 file changed, 3 insertions(+), 3 deletions(-) diff --git a/docs/usage.md b/docs/usage.md index aab4a249..a95cb1d8 100644 --- a/docs/usage.md +++ b/docs/usage.md @@ -11,7 +11,7 @@ * [`-profile`](#-profile) * [`--input`](#--input) * [`--protocol`](#--protocol) -* [Basecalling/Demultiplexing](#basecalling-demultiplexing) +* [Basecalling and demultiplexing](#basecalling-and-demultiplexing) * [`--input_path`](#--input_path) * [`--flowcell`](#--flowcell) * [`--kit`](#--kit) @@ -267,12 +267,12 @@ nextflow run nf-core/nanoseq \ Specifies the type of data that was sequenced i.e. "DNA", "cDNA" or "directRNA". +## Basecalling and demultiplexing + ### `--input_path` Path to Nanopore run directory (e.g. `fastq_pass/`) or a basecalled fastq file that requires demultiplexing. The latter can only be provided in conjunction with the `--skip_basecalling` parameter. -## Basecalling/Demultiplexing - ### `--flowcell` Flowcell used to perform the sequencing e.g. "FLO-MIN106". Not required if `--guppy_config` is specified. From 13bf1db096330a4bc318cf6672c150fbf83b672d Mon Sep 17 00:00:00 2001 From: drpatelh Date: Wed, 12 Feb 2020 17:15:32 +0000 Subject: [PATCH 08/22] Update docs --- docs/usage.md | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/docs/usage.md b/docs/usage.md index a95cb1d8..d51ec9c3 100644 --- a/docs/usage.md +++ b/docs/usage.md @@ -341,11 +341,11 @@ Search for adapters in the whole read by applying the '--detect-middle' paramete ### `--skip_basecalling` -Skip basecalling with Guppy +Skip basecalling with Guppy. ### `--skip_demultiplexing` -Skip demultiplexing with Guppy/qcat +Skip demultiplexing with Guppy/qcat. ## Alignment From 9bbc9193d65b2dde1c2dc3e7dde8300dfae69b17 Mon Sep 17 00:00:00 2001 From: drpatelh Date: Wed, 12 Feb 2020 17:19:37 +0000 Subject: [PATCH 09/22] Update descriptions --- main.nf | 8 ++++---- 1 file changed, 4 insertions(+), 4 deletions(-) diff --git a/main.nf b/main.nf index eda5290c..10133a8c 100644 --- a/main.nf +++ b/main.nf @@ -40,8 +40,8 @@ def helpMessage() { --guppy_config [file/str] Guppy config file used for basecalling. Cannot be used in conjunction with '--flowcell' and '--kit' --guppy_model [file/str] Custom basecalling model file (JSON) to use for Guppy basecalling, such as the output from Taiyaki (Default: false) --guppy_gpu [bool] Whether to perform basecalling with Guppy in GPU mode (Default: false) - --guppy_gpu_runners [int] Number of '--gpu_runners_per_device' used for guppy when using '--guppy_gpu' (Default: 6) - --guppy_cpu_threads [int] Number of '--cpu_threads_per_caller' used for guppy when using '--guppy_gpu' (Default: 1) + --guppy_gpu_runners [int] Number of '--gpu_runners_per_device' used for Guppy when using '--guppy_gpu' (Default: 6) + --guppy_cpu_threads [int] Number of '--cpu_threads_per_caller' used for Guppy when using '--guppy_gpu' (Default: 1) --gpu_device [str] Basecalling device specified to Guppy in GPU mode using '--device' (Default: 'auto') --gpu_cluster_options [str] Cluster options required to use GPU resources (e.g. '--part=gpu --gres=gpu:1') --qcat_min_score [int] Minimum scores of '--min-score' used for qcat (Default: 60) @@ -51,8 +51,8 @@ def helpMessage() { Alignment --aligner [str] Specifies the aligner to use (available are: minimap2 or graphmap2) (Default: 'minimap2') - --stranded [bool] Specifies if the data is strand-specific. Automatically activated when using --protocol directRNA (Default: false) - --save_align_intermeds [bool] Save the .sam files from the alignment step (Default: false) + --stranded [bool] Specifies if the data is strand-specific. Automatically activated when using '--protocol directRNA' (Default: false) + --save_align_intermeds [bool] Save the '.sam' files from the alignment step (Default: false) --skip_alignment [bool] Skip alignment and subsequent process (Default: false) Coverage tracks From 893350bb13202fc07b5fa056bc915e65bdae10ba Mon Sep 17 00:00:00 2001 From: drpatelh Date: Thu, 13 Feb 2020 14:00:07 +0000 Subject: [PATCH 10/22] Update output docs --- docs/output.md | 28 +++++++++++++++------------- 1 file changed, 15 insertions(+), 13 deletions(-) diff --git a/docs/output.md b/docs/output.md index cb5441e0..52c2e2c5 100644 --- a/docs/output.md +++ b/docs/output.md @@ -2,28 +2,30 @@ This document describes the output produced by the pipeline. Most of the plots are taken from the MultiQC report, which summarises results at the end of the pipeline. - - ## Pipeline overview -The pipeline is built using [Nextflow](https://www.nextflow.io/) and processes data using the following steps: +The pipeline is built using [Nextflow](https://www.nextflow.io/). See [`main README.md`](../README.md) for a condensed overview of the steps in the pipeline, and the bioinformatics tools used at each step. + +See [Oxford NanoPore website](https://nanoporetech.com/) for more information regarding the sequencing technology, protocol, and for an extensive list of resources. -* [Guppy](#guppy) - demultiplexing of Nanopore data -* [PycoQC](#pycoqc) - read quality control -* [NanoPlot](#nanoplot) - read quality control -* [GraphMap2](#graphmap2) - mapping for long reads -* [MiniMap2](#minimap2) - mapping for long reads -* [SortBam](#sortbam) - coordinate sort BAM files using SAMtools -* [bedtools](#bedtools) - create bigWig and bigBed files -* [MultiQC](#multiqc) - aggregate report, describing results of the alignment +The directories listed below will be created in the output directory after the pipeline has finished. All paths are relative to the top-level results directory. -## Demultiplexing +## Basecalling and demultiplexing *Documentation*: -[Guppy](https://nanoporetech.com/nanopore-sequencing-data-analysis) +[Guppy](https://nanoporetech.com/nanopore-sequencing-data-analysis), [qcat](https://github.com/nanoporetech/qcat) *Description*: +The pipeline has been written to deal with the various scenarios where you would like to include/exclude the basecalling and demultiplexing steps. This will be dependent on what type of input data you would like to provide the pipeline. Additionally, if you would like to align your samples to a reference genome there are various options for providing this information. Please see [`usage.md`](usage.md#--input) for more details about the format of the input samplesheet, associated commands and how to provide reference genome data. + +Guppy will be used to basecall and demultiplex the data. However, if you have a pre-basecalled fastq file then qcat will be used to perform the demultiplexing if you provide the `--skip_basecalling` parameter. If you would like to skip both of these steps entirely then you can provide `--skip_basecalling --skip_demultiplexing` when running the pipeline. As a result, the structure of the output folder will depend on which steps you have chosen to run in the pipeline. + +Assuming you would like to perform both basecalling and demultiplexing the + + Guppy will demultiplex and barcode the data given from an ONT device. The flowcell, kit and barcode kit must be given in the command line if demultiplexing needed. This step can by bypassed using the `--skip_demultiplexing` parameter when initiating the pipeline. The output folders will be separated into the barcodes from the kit used and unclassified. The output in each barcode folder is then merged into one fastq file for easier downstream processing. +Pipeline provides various parameters to customise these parameters. +Guppy can also use GPU resources if you have them available *Output directories*: From 4d04cfcc83ced8fb35e03998d18e8c5fd23c46c5 Mon Sep 17 00:00:00 2001 From: drpatelh Date: Thu, 13 Feb 2020 15:01:49 +0000 Subject: [PATCH 11/22] Update output docs --- docs/output.md | 27 ++++++++++++++++----------- 1 file changed, 16 insertions(+), 11 deletions(-) diff --git a/docs/output.md b/docs/output.md index 52c2e2c5..32aa6b06 100644 --- a/docs/output.md +++ b/docs/output.md @@ -20,21 +20,26 @@ The pipeline has been written to deal with the various scenarios where you would Guppy will be used to basecall and demultiplex the data. However, if you have a pre-basecalled fastq file then qcat will be used to perform the demultiplexing if you provide the `--skip_basecalling` parameter. If you would like to skip both of these steps entirely then you can provide `--skip_basecalling --skip_demultiplexing` when running the pipeline. As a result, the structure of the output folder will depend on which steps you have chosen to run in the pipeline. -Assuming you would like to perform both basecalling and demultiplexing the - - -Guppy will demultiplex and barcode the data given from an ONT device. The flowcell, kit and barcode kit must be given in the command line if demultiplexing needed. This step can by bypassed using the `--skip_demultiplexing` parameter when initiating the pipeline. The output folders will be separated into the barcodes from the kit used and unclassified. The output in each barcode folder is then merged into one fastq file for easier downstream processing. -Pipeline provides various parameters to customise these parameters. -Guppy can also use GPU resources if you have them available +Assuming you would like to perform both basecalling and demultiplexing: *Output directories*: -* `guppy/basecalling/barcode*/` - FastQ files output for each barcode -* `guppy/basecalling/unclassified/` - FastQ files output that are unclassified * `guppy/fastq/` - Merged output of fastq files into one fastq for each barcode + Merged FastQ output files for each barcode +* `guppy/basecalling//` + FastQ output files for each barcode +* `guppy/basecalling/unclassified/` + FastQ files with reads were unassigned to any given barcode +* `guppy/basecalling/sequencing_summary.txt` + Sequencing summary file generated by Guppy +* `guppy/basecalling/sequencing_telemetry.js` + Sequencing telemetry file generated by Guppy +* `guppy/basecalling/guppy_basecaller_log-.log` + Log file for Guppy execution + + + Pipeline provides various parameters to customise these parameters. + Guppy can also use GPU resources if you have them available ## Sequencing Quality Control From 22b6e94c2a78b5e351c7fafe0c51e3ca5148b4a9 Mon Sep 17 00:00:00 2001 From: drpatelh Date: Thu, 13 Feb 2020 15:01:58 +0000 Subject: [PATCH 12/22] Change version string --- main.nf | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/main.nf b/main.nf index 10133a8c..d9c990e4 100644 --- a/main.nf +++ b/main.nf @@ -312,7 +312,7 @@ if (!params.skip_basecalling) { label 'process_high' publishDir path: "${params.outdir}/guppy", mode: 'copy', saveAs: { filename -> - if (!filename.endsWith(".version")) filename + if (!filename.endsWith("guppy.txt")) filename } input: From 2768967f865f460dbbff7c9c110a962f7c74d363 Mon Sep 17 00:00:00 2001 From: drpatelh Date: Thu, 13 Feb 2020 16:21:34 +0000 Subject: [PATCH 13/22] Update docs --- docs/output.md | 25 +++++++++++++------------ 1 file changed, 13 insertions(+), 12 deletions(-) diff --git a/docs/output.md b/docs/output.md index 32aa6b06..140646f2 100644 --- a/docs/output.md +++ b/docs/output.md @@ -18,28 +18,28 @@ The directories listed below will be created in the output directory after the p *Description*: The pipeline has been written to deal with the various scenarios where you would like to include/exclude the basecalling and demultiplexing steps. This will be dependent on what type of input data you would like to provide the pipeline. Additionally, if you would like to align your samples to a reference genome there are various options for providing this information. Please see [`usage.md`](usage.md#--input) for more details about the format of the input samplesheet, associated commands and how to provide reference genome data. -Guppy will be used to basecall and demultiplex the data. However, if you have a pre-basecalled fastq file then qcat will be used to perform the demultiplexing if you provide the `--skip_basecalling` parameter. If you would like to skip both of these steps entirely then you can provide `--skip_basecalling --skip_demultiplexing` when running the pipeline. As a result, the structure of the output folder will depend on which steps you have chosen to run in the pipeline. +Guppy will be used to basecall and demultiplex the data. Various options have been provided to customise specific parameters and to be able to run Guppy on GPUs. -Assuming you would like to perform both basecalling and demultiplexing: +If you have a pre-basecalled fastq file then qcat will be used to perform the demultiplexing if you provide the `--skip_basecalling` parameter. If you would like to skip both of these steps entirely then you can provide `--skip_basecalling --skip_demultiplexing` when running the pipeline. As a result, the structure of the output folder will depend on which steps you have chosen to run in the pipeline. *Output directories*: * `guppy/fastq/` - Merged FastQ output files for each barcode + Merged FastQ output files for each barcode. * `guppy/basecalling//` - FastQ output files for each barcode + FastQ output files for each barcode. * `guppy/basecalling/unclassified/` - FastQ files with reads were unassigned to any given barcode + FastQ files with reads were unassigned to any given barcode. * `guppy/basecalling/sequencing_summary.txt` - Sequencing summary file generated by Guppy + Sequencing summary file generated by Guppy. * `guppy/basecalling/sequencing_telemetry.js` - Sequencing telemetry file generated by Guppy + Sequencing telemetry file generated by Guppy. * `guppy/basecalling/guppy_basecaller_log-.log` - Log file for Guppy execution - - - Pipeline provides various parameters to customise these parameters. - Guppy can also use GPU resources if you have them available + Log file for Guppy execution. +* `qcat/fastq/.fastq.gz` + FastQ output files for each barcode. +* `qcat/fastq/none.fastq.gz` + FastQ file with reads were unassigned to any given barcode. ## Sequencing Quality Control @@ -48,6 +48,7 @@ Assuming you would like to perform both basecalling and demultiplexing: *Description*: PycoQC and NanoPlot give general quality metrics about the sequencing run. It provides information about the distribution of read length, read length over time, number of reads per barcode and other general stats. + ![PycoQC - Number of Reads per Barcode plot](images/NumberofReadsperBarcode.png) *Output directories*: From c38a07aa86f885b115620ef42b5ee16ea1ebd417 Mon Sep 17 00:00:00 2001 From: drpatelh Date: Fri, 14 Feb 2020 12:25:06 +0000 Subject: [PATCH 14/22] Update usage docs --- docs/output.md | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/docs/output.md b/docs/output.md index 140646f2..938d4ba7 100644 --- a/docs/output.md +++ b/docs/output.md @@ -41,13 +41,13 @@ If you have a pre-basecalled fastq file then qcat will be used to perform the de * `qcat/fastq/none.fastq.gz` FastQ file with reads were unassigned to any given barcode. -## Sequencing Quality Control +## Sequencing QC *Documentation*: [PycoQC](https://github.com/a-slide/pycoQC), [NanoPlot](https://github.com/wdecoster/NanoPlot) *Description*: -PycoQC and NanoPlot give general quality metrics about the sequencing run. It provides information about the distribution of read length, read length over time, number of reads per barcode and other general stats. +PycoQC and NanoPlot compute metrics and generate QC plots using the sequencing summary information generated by Guppy e.g. distribution of read length, read length over time, number of reads per barcode and other general stats. NanoPlot also generates QC metrics directly from FastQ files. ![PycoQC - Number of Reads per Barcode plot](images/NumberofReadsperBarcode.png) From b80156981c1e0d70b7da80509ae513296bee1ea5 Mon Sep 17 00:00:00 2001 From: drpatelh Date: Fri, 14 Feb 2020 12:59:32 +0000 Subject: [PATCH 15/22] Update images --- docs/images/NumberofReadsperBarcode.png | Bin 77733 -> 0 bytes docs/images/SampleHistogramReadlength.png | Bin 20023 -> 0 bytes ...plot_1.png => mqc_samtools_stats_plot.png} | Bin ...put.png => nanoplot_readlengthquality.png} | Bin docs/images/pycoQC_output.html | 363 ------------------ docs/images/pycoqc_readsperbarcode.png | Bin 0 -> 24100 bytes docs/output.md | 6 +- 7 files changed, 3 insertions(+), 366 deletions(-) delete mode 100644 docs/images/NumberofReadsperBarcode.png delete mode 100644 docs/images/SampleHistogramReadlength.png rename docs/images/{mqc_samtools_alignment_plot_1.png => mqc_samtools_stats_plot.png} (100%) rename docs/images/{NanoPlot_output.png => 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    PycoQC report

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    Generate on 19/08/19 with pycoQC 2.2.3.7

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    Run summary

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    Run summary by barcode

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    Run summary by Run ID

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    Distribution of read length

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    Distribution of read quality

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    Mean read quality per sequence length

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    Output over experiment time

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    Read length over time

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    Mean read quality over time

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    Number of reads per barcode

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    Channel activity over time

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zi{k4U^>c3?Gq`-xWC4S)`7RSEtnr%BFaMfq&yP4UTk1Q@Kn_@~hE4ehK&fopy3=SX z$a0IUp%E-4Y+<$uuID4-_eR59$t_tj7kNVttR<|>kXewLKKwzt{pF%XIdMkLT_Tor zzSlxv?Xd2FBdJ+2OdhSbzne%@q77f(9HAxkb}tb2VXuWM9c{4!SF>mvKG`Haw&9rKu4CVJsGme3L*wwb zRf(Xkvzqv8IGba2NU&Xwvud!Dy6rBN50NEsIxyl(yzwiY)cZ#dnKzf(LrNWXor=}J zE=cRg6Ip?61q5bX>w}5{af!z)_mwerRM<38 zE!Lh*75WKh2Z*= zNJ!B!L94|rT3F*30&WLvS(`XjXJ& zcN3m!b^2->pVa2ey-_T)di{mICvWgkC+~iSBKvm68=sM_yEbdaY57!6!J$x1SXd-$ zf_-@RFn39v%fybl&4XlN;n?}#HbKML8;@IV?1xG=XawNRw|xs12KQ69*;#m_2-EKc zodYDrlnPpmvsMkQ9+lON6n3wD{B1U{{PuH=jgC-M)Wz;>^HZ+@f|Q2rlqET Kt5nrG Date: Fri, 14 Feb 2020 13:04:46 +0000 Subject: [PATCH 16/22] Update image path --- docs/output.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/docs/output.md b/docs/output.md index 4fdf3591..66b85bc6 100644 --- a/docs/output.md +++ b/docs/output.md @@ -49,7 +49,7 @@ If you have a pre-basecalled fastq file then qcat will be used to perform the de *Description*: PycoQC and NanoPlot compute metrics and generate QC plots using the sequencing summary information generated by Guppy e.g. distribution of read length, read length over time, number of reads per barcode and other general stats. NanoPlot also generates QC metrics directly from FastQ files. -![PycoQC - Number of reads per barcode](images/test.png) +![PycoQC - Number of reads per barcode](images/pycoqc_readsperbarcode.png) *Output directories*: From 1dd28ccb8c5a2ab40b9d602d976f8ec215841456 Mon Sep 17 00:00:00 2001 From: drpatelh Date: Fri, 14 Feb 2020 13:25:16 +0000 Subject: [PATCH 17/22] Update environment --- docs/output.md | 19 ++++++++++++------- 1 file changed, 12 insertions(+), 7 deletions(-) diff --git a/docs/output.md b/docs/output.md index 66b85bc6..688e1753 100644 --- a/docs/output.md +++ b/docs/output.md @@ -53,24 +53,29 @@ PycoQC and NanoPlot compute metrics and generate QC plots using the sequencing s *Output directories*: -* `pycoQC/` - An .html file output is produced that includes a run summary and graphical representation of distribution of read length, distribution of read quality scores, mean read quality per sequence length, output per channel over experiment time, output over experiment time, read quality over experiment time, readlength over experiment time, and percentage of reads per barcode. +* `pycoqc/pycoQC_output.html` + `*.html` file that includes a run summary and graphical representation of various QC metrics including distribution of read length, distribution of read quality scores, mean read quality per sequence length, output per channel over experiment time and percentage of reads per barcode. * `nanoplot/summary/` - An output of .png files of metric plots and an html summary file of overall run. + `*.html` files for QC metrics and individual `*.png` image files for plots. -## FastQ Quality Control +## FastQ QC *Documentation*: -[NanoPlot](https://github.com/wdecoster/NanoPlot) +[NanoPlot](https://github.com/wdecoster/NanoPlot), [FastQC](http://www.bioinformatics.babraham.ac.uk/projects/fastqc/Help/) *Description*: NanoPlot give general quality metrics about the fastq output per barcode from Guppy. It provides information about the quality score distribution across your reads, read lengths and other general stats. + ![Nanoplot - Read quality vs read length](images/nanoplot_readlengthquality.png) +FastQC gives general quality metrics about your reads. It provides information about the quality score distribution across your reads, the per base sequence content (%A/C/G/T). You get information about adapter contamination and other overrepresented sequences. + *Output directories*: -* `nanoplot/fastq/` - An output of QC metric plots in individual .png files and in one html file summarizing the output. +* `nanoplot/fastq//` + Per sample `*.html` files for QC metrics and individual `*.png` image files for plots. +* `fastqc/` + FastQC `*.html` and `*.zip` files. ## Alignment From 8f59acc7802f55d0ad51d2a5df102026e40d79ca Mon Sep 17 00:00:00 2001 From: drpatelh Date: Fri, 14 Feb 2020 13:57:27 +0000 Subject: [PATCH 18/22] Update environment --- docs/output.md | 58 +++++++++++++++++++++++++------------------------ environment.yml | 1 + 2 files changed, 31 insertions(+), 28 deletions(-) diff --git a/docs/output.md b/docs/output.md index 688e1753..994be1d1 100644 --- a/docs/output.md +++ b/docs/output.md @@ -73,62 +73,65 @@ FastQC gives general quality metrics about your reads. It provides information a *Output directories*: * `nanoplot/fastq//` - Per sample `*.html` files for QC metrics and individual `*.png` image files for plots. + Per-sample `*.html` files for QC metrics and individual `*.png` image files for plots. * `fastqc/` FastQC `*.html` and `*.zip` files. ## Alignment *Documentation*: -[GraphMap2](https://github.com/lbcb-sci/graphmap2), [MiniMap2](https://github.com/lh3/minimap2), [SortBam](http://www.htslib.org/doc/samtools.html) +[GraphMap2](https://github.com/lbcb-sci/graphmap2), [MiniMap2](https://github.com/lh3/minimap2), [SAMtools](http://samtools.sourceforge.net/) *Description*: -The FastQ reads are mapped to the given reference assembly provided using either GraphMap2 or Minimap2 and then sorted and indexed using SAMtools or these processes can be bypassed using the `--skip_alignment` parameter. +Reads are mapped to a user-defined genome or transcriptome using either GraphMap2 or Minimap2, and the resulting BAM files are sorted and indexed. If the same reference is specified multiple times in the input samplesheet then the aligner index will only be built once for re-use across all samples. You can skip the alignment and downstream processes by providing the `--skip_alignment` parameter. -The files resulting from the alignment with graphmap2 or minimap2 of individual libraries are not saved by default so this directory will not be present in your results. You can override this behaviour with the use of the `--save_align_intermeds` flag in which case it will contain the coordinate sorted alignment files in [`*.bam`](https://samtools.github.io/hts-specs/SAMv1.pdf) format. +The initial SAM alignment files created by GraphMap2 or Minimap2 are not saved by default to be more efficient with storage space. You can override this behaviour with the use of the `--save_align_intermeds` flag. ![MultiQC - SAMtools stats plot](images/mqc_samtools_stats_plot.png) *Output directories*: -* `graphmap2/` - If the `--aligner graphmap2` parameter is used, the sorted and indexed bam files will be output here. -* `minimap2/` - If the `--aligner minimap2` parameter is used, the sorted and indexed bam files will be output here. -* `/samtools_stats/` - `*.flagstat`, `*.idxstats` and `*.stats` files generated from the alignment files using SAMtools. +* `/` + Per-sample coordinate sorted alignment files in [`*.bam`](https://samtools.github.io/hts-specs/SAMv1.pdf) format. +* `/samtools_stats/` + SAMtools `*.flagstat`, `*.idxstats` and `*.stats` files generated from the alignment files. -## bigWig and bigBed +## Coverage tracks *Documentation*: -[`BEDTools`](https://github.com/arq5x/bedtools2/), [`bedGraphToBigWig`](http://hgdownload.soe.ucsc.edu/admin/exe/), [`bedToBigBed`](http://hgdownload.soe.ucsc.edu/admin/exe/) +[BEDTools](https://bedtools.readthedocs.io/en/latest/), [bedGraphToBigWig](https://genome.ucsc.edu/goldenpath/help/bigWig.html#Ex3), [`bedToBigBed`](https://genome.ucsc.edu/goldenPath/help/bigBed.html#Ex2) *Description*: -Creation of bigWig and bigBed coverage tracks for visualisation. This can be bypassed by setting the parameters `--skip_bigwig` and/or `--skip_bigbed`. +The [bigWig](https://genome.ucsc.edu/goldenpath/help/bigWig.html) format is in an indexed binary format useful for displaying dense, continuous data in Genome Browsers such as the [UCSC](https://genome.ucsc.edu/cgi-bin/hgTracks) and [IGV](http://software.broadinstitute.org/software/igv/). This mitigates the need to load the much larger BAM files for data visualisation purposes which will be slower and result in memory issues. The bigWig format is also supported by various bioinformatics software for downstream processing such as meta-profile plotting. + +[bigBed](https://genome.ucsc.edu/goldenPath/help/bigBed.html) are more useful for displaying distribution of reads across exon intervals as is typically observed for RNA-seq data. Therefore, these files will only be generated if `--protocol directRNA` or `--protocol cDNA` are defined. + +The creation of these files can be bypassed by setting the parameters `--skip_bigwig`/`--skip_bigbed`. *Output directories*: * `/bigwig/` - The bigWig files will be output here. -* `/bigbed/` - The bigbed files will be output here. + Per-sample `*.bigWig` files. +* `/bigbed/` + Per-sample `*.bigBed` files. ## MultiQC -[MultiQC](http://multiqc.info) is a visualisation tool that generates a single HTML report summarising all samples in your project. Most of the pipeline QC results are visualised in the report and further statistics are available in within the report data directory. +*Documentation*: +[MultiQC](https://multiqc.info/docs/) -The pipeline has special steps which allow the software versions used to be reported in the MultiQC output for future traceability. +*Description*: +MultiQC is a visualisation tool that generates a single HTML report summarising all samples in your project. Most of the pipeline QC results are visualised in the report and further statistics are available within the report data directory. -*Output directories*: +Results generated by MultiQC for this pipeline collate QC from FastQC, samtools flagstat, samtools idxstats and samtools stats. -* `multiqc/Project_multiqc_report.html` - MultiQC report - a standalone HTML file that can be viewed in your web browser -* `multiqc/multiqc_data/` - Directory containing parsed statistics from the different tools used in the pipeline -* `multiqc/multiqc_plots/` - Directory containing the image files of the graphs included in MultiQC +The pipeline has special steps which also allow the software versions to be reported in the MultiQC output for future traceability. For more information about how to use MultiQC reports, see . -For more information about how to use MultiQC reports, see [http://multiqc.info](http://multiqc.info) +*Output directories*: +* `multiqc/` + * `multiqc_report.html` - a standalone HTML file that can be viewed in your web browser. + * `multiqc_data/` - directory containing parsed statistics from the different tools used in the pipeline. + * `multiqc_plots/` - directory containing static images from the report in various formats. ## Pipeline information @@ -144,5 +147,4 @@ Nextflow provides excellent functionality for generating various reports relevan * Reports generated by the pipeline - `pipeline_report.html`, `pipeline_report.txt` and `software_versions.csv`. * Reports generated by Nextflow - `execution_report.html`, `execution_timeline.html`, `execution_trace.txt` and `pipeline_dag.svg`. * Reformatted samplesheet files used as input to the pipeline - `samplesheet_reformat.csv`. -* `Documentation/` - Documentation for interpretation of results in HTML format - `results_description.html`. + * Documentation for interpretation of results in HTML format - `results_description.html`. diff --git a/environment.yml b/environment.yml index 31ec140a..4681d068 100644 --- a/environment.yml +++ b/environment.yml @@ -11,6 +11,7 @@ dependencies: - conda-forge::pymdown-extensions=6.0 - conda-forge::pygments=2.5.2 - conda-forge::pigz=2.3.4 + - conda-forge::psutil=5.6.7 - bioconda::fastqc=0.11.9 - bioconda::multiqc=1.8 From f04140ca19e090c7d7b9f945e73ae5e1ef419134 Mon Sep 17 00:00:00 2001 From: drpatelh Date: Fri, 14 Feb 2020 15:10:28 +0000 Subject: [PATCH 19/22] Italicise tools --- docs/output.md | 46 +++++++++++++++++++++++----------------------- 1 file changed, 23 insertions(+), 23 deletions(-) diff --git a/docs/output.md b/docs/output.md index 994be1d1..e1f137cb 100644 --- a/docs/output.md +++ b/docs/output.md @@ -18,28 +18,28 @@ The directories listed below will be created in the output directory after the p *Description*: The pipeline has been written to deal with the various scenarios where you would like to include/exclude the basecalling and demultiplexing steps. This will be dependent on what type of input data you would like to provide the pipeline. Additionally, if you would like to align your samples to a reference genome there are various options for providing this information. Please see [`usage.md`](usage.md#--input) for more details about the format of the input samplesheet, associated commands and how to provide reference genome data. -Guppy will be used to basecall and demultiplex the data. Various options have been provided to customise specific parameters and to be able to run Guppy on GPUs. +*Guppy* will be used to basecall and demultiplex the data. Various options have been provided to customise specific parameters and to be able to run *Guppy* on GPUs. -If you have a pre-basecalled fastq file then qcat will be used to perform the demultiplexing if you provide the `--skip_basecalling` parameter. If you would like to skip both of these steps entirely then you can provide `--skip_basecalling --skip_demultiplexing` when running the pipeline. As a result, the structure of the output folder will depend on which steps you have chosen to run in the pipeline. +If you have a pre-basecalled fastq file then *qcat* will be used to perform the demultiplexing if you provide the `--skip_basecalling` parameter. If you would like to skip both of these steps entirely then you can provide `--skip_basecalling --skip_demultiplexing` when running the pipeline. As a result, the structure of the output folder will depend on which steps you have chosen to run in the pipeline. *Output directories*: * `guppy/fastq/` - Merged FastQ output files for each barcode. + Merged fastq output files for each barcode. * `guppy/basecalling//` - FastQ output files for each barcode. + fastq output files for each barcode. * `guppy/basecalling/unclassified/` - FastQ files with reads were unassigned to any given barcode. + fastq files with reads were unassigned to any given barcode. * `guppy/basecalling/sequencing_summary.txt` - Sequencing summary file generated by Guppy. + Sequencing summary file generated by *Guppy*. * `guppy/basecalling/sequencing_telemetry.js` - Sequencing telemetry file generated by Guppy. + Sequencing telemetry file generated by *Guppy*. * `guppy/basecalling/guppy_basecaller_log-.log` - Log file for Guppy execution. + Log file for *Guppy* execution. * `qcat/fastq/.fastq.gz` - FastQ output files for each barcode. + fastq output files for each barcode. * `qcat/fastq/none.fastq.gz` - FastQ file with reads were unassigned to any given barcode. + fastq file with reads were unassigned to any given barcode. ## Sequencing QC @@ -47,7 +47,7 @@ If you have a pre-basecalled fastq file then qcat will be used to perform the de [PycoQC](https://github.com/a-slide/pycoQC), [NanoPlot](https://github.com/wdecoster/NanoPlot) *Description*: -PycoQC and NanoPlot compute metrics and generate QC plots using the sequencing summary information generated by Guppy e.g. distribution of read length, read length over time, number of reads per barcode and other general stats. NanoPlot also generates QC metrics directly from FastQ files. +*PycoQC* and *NanoPlot* compute metrics and generate QC plots using the sequencing summary information generated by *Guppy* e.g. distribution of read length, read length over time, number of reads per barcode and other general stats. *NanoPlot* also generates QC metrics directly from fastq files as described in the next section. ![PycoQC - Number of reads per barcode](images/pycoqc_readsperbarcode.png) @@ -64,18 +64,18 @@ PycoQC and NanoPlot compute metrics and generate QC plots using the sequencing s [NanoPlot](https://github.com/wdecoster/NanoPlot), [FastQC](http://www.bioinformatics.babraham.ac.uk/projects/fastqc/Help/) *Description*: -NanoPlot give general quality metrics about the fastq output per barcode from Guppy. It provides information about the quality score distribution across your reads, read lengths and other general stats. +*NanoPlot* give general quality metrics about the fastq output per barcode from *Guppy*. It provides information about the quality score distribution across your reads, read lengths and other general stats. ![Nanoplot - Read quality vs read length](images/nanoplot_readlengthquality.png) -FastQC gives general quality metrics about your reads. It provides information about the quality score distribution across your reads, the per base sequence content (%A/C/G/T). You get information about adapter contamination and other overrepresented sequences. +*FastQC* gives general quality metrics about your reads. It provides information about the quality score distribution across your reads, the per base sequence content (%A/C/G/T). You get information about adapter contamination and other overrepresented sequences. *Output directories*: * `nanoplot/fastq//` Per-sample `*.html` files for QC metrics and individual `*.png` image files for plots. * `fastqc/` - FastQC `*.html` and `*.zip` files. + *FastQC* `*.html` and `*.zip` files. ## Alignment @@ -83,9 +83,9 @@ FastQC gives general quality metrics about your reads. It provides information a [GraphMap2](https://github.com/lbcb-sci/graphmap2), [MiniMap2](https://github.com/lh3/minimap2), [SAMtools](http://samtools.sourceforge.net/) *Description*: -Reads are mapped to a user-defined genome or transcriptome using either GraphMap2 or Minimap2, and the resulting BAM files are sorted and indexed. If the same reference is specified multiple times in the input samplesheet then the aligner index will only be built once for re-use across all samples. You can skip the alignment and downstream processes by providing the `--skip_alignment` parameter. +Reads are mapped to a user-defined genome or transcriptome using either *GraphMap2* or *Minimap2*, and the resulting BAM files are sorted and indexed. If the same reference is specified multiple times in the input sample sheet then the aligner index will only be built once for re-use across all samples. You can skip the alignment and downstream processes by providing the `--skip_alignment` parameter. -The initial SAM alignment files created by GraphMap2 or Minimap2 are not saved by default to be more efficient with storage space. You can override this behaviour with the use of the `--save_align_intermeds` flag. +The initial SAM alignment files created by *GraphMap2* or *Minimap2* are not saved by default to be more efficient with storage space. You can override this behaviour with the use of the `--save_align_intermeds` flag. ![MultiQC - SAMtools stats plot](images/mqc_samtools_stats_plot.png) @@ -94,7 +94,7 @@ The initial SAM alignment files created by GraphMap2 or Minimap2 are not saved b * `/` Per-sample coordinate sorted alignment files in [`*.bam`](https://samtools.github.io/hts-specs/SAMv1.pdf) format. * `/samtools_stats/` - SAMtools `*.flagstat`, `*.idxstats` and `*.stats` files generated from the alignment files. + *SAMtools* `*.flagstat`, `*.idxstats` and `*.stats` files generated from the alignment files. ## Coverage tracks @@ -121,11 +121,11 @@ The creation of these files can be bypassed by setting the parameters `--skip_bi [MultiQC](https://multiqc.info/docs/) *Description*: -MultiQC is a visualisation tool that generates a single HTML report summarising all samples in your project. Most of the pipeline QC results are visualised in the report and further statistics are available within the report data directory. +*MultiQC* is a visualisation tool that generates a single HTML report summarising all samples in your project. Most of the pipeline QC results are visualised in the report and further statistics are available within the report data directory. -Results generated by MultiQC for this pipeline collate QC from FastQC, samtools flagstat, samtools idxstats and samtools stats. +Results generated by *MultiQC* for this pipeline collate QC from *FastQC*, *samtools flagstat*, *samtools idxstats* and *samtools stats*. -The pipeline has special steps which also allow the software versions to be reported in the MultiQC output for future traceability. For more information about how to use MultiQC reports, see . +The pipeline has special steps which also allow the software versions to be reported in the *MultiQC* output for future traceability. For more information about how to use *MultiQC* reports, see . *Output directories*: * `multiqc/` @@ -139,12 +139,12 @@ The pipeline has special steps which also allow the software versions to be repo [Nextflow](https://www.nextflow.io/docs/latest/tracing.html) *Description*: -Nextflow provides excellent functionality for generating various reports relevant to the running and execution of the pipeline. This will allow you to trouble-shoot errors with the running of the pipeline, and also provide you with other information such as launch commands, run times and resource usage. +*Nextflow* provides excellent functionality for generating various reports relevant to the running and execution of the pipeline. This will allow you to trouble-shoot errors with the running of the pipeline, and also provide you with other information such as launch commands, run times and resource usage. *Output directories*: * `pipeline_info/` * Reports generated by the pipeline - `pipeline_report.html`, `pipeline_report.txt` and `software_versions.csv`. - * Reports generated by Nextflow - `execution_report.html`, `execution_timeline.html`, `execution_trace.txt` and `pipeline_dag.svg`. - * Reformatted samplesheet files used as input to the pipeline - `samplesheet_reformat.csv`. + * Reports generated by *Nextflow* - `execution_report.html`, `execution_timeline.html`, `execution_trace.txt` and `pipeline_dag.svg`. + * Reformatted sample sheet files used as input to the pipeline - `samplesheet_reformat.csv`. * Documentation for interpretation of results in HTML format - `results_description.html`. 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-*NanoPlot* give general quality metrics about the fastq output per barcode from *Guppy*. It provides information about the quality score distribution across your reads, read lengths and other general stats. +*NanoPlot* can also be used to produce general quality metrics from the per-barcode fastq files generated by *Guppy* e.g. quality score distribution, read lengths and other general stats. ![Nanoplot - Read quality vs read length](images/nanoplot_readlengthquality.png) @@ -72,7 +72,7 @@ If you have a pre-basecalled fastq file then *qcat* will be used to perform the *Output directories*: -* `nanoplot/fastq//` +* `nanoplot/fastq//` Per-sample `*.html` files for QC metrics and individual `*.png` image files for plots. * `fastqc/` *FastQC* `*.html` and `*.zip` files. @@ -85,7 +85,7 @@ If you have a pre-basecalled fastq file then *qcat* will be used to perform the *Description*: Reads are mapped to a user-defined genome or transcriptome using either *GraphMap2* or *Minimap2*, and the resulting BAM files are sorted and indexed. If the same reference is specified multiple times in the input sample sheet then the aligner index will only be built once for re-use across all samples. You can skip the alignment and downstream processes by providing the `--skip_alignment` parameter. -The initial SAM alignment files created by *GraphMap2* or *Minimap2* are not saved by default to be more efficient with storage space. You can override this behaviour with the use of the `--save_align_intermeds` flag. +The initial SAM alignment files created by *GraphMap2* or *Minimap2* are not saved by default to be more efficient with storage space. You can override this behaviour with the use of the `--save_align_intermeds` parameter. ![MultiQC - SAMtools stats plot](images/mqc_samtools_stats_plot.png) @@ -98,10 +98,10 @@ The initial SAM alignment files created by *GraphMap2* or *Minimap2* are not sav ## Coverage tracks -*Documentation*: +*Documentation*: [BEDTools](https://bedtools.readthedocs.io/en/latest/), [bedGraphToBigWig](https://genome.ucsc.edu/goldenpath/help/bigWig.html#Ex3), [`bedToBigBed`](https://genome.ucsc.edu/goldenPath/help/bigBed.html#Ex2) -*Description*: +*Description*: The [bigWig](https://genome.ucsc.edu/goldenpath/help/bigWig.html) format is in an indexed binary format useful for displaying dense, continuous data in Genome Browsers such as the [UCSC](https://genome.ucsc.edu/cgi-bin/hgTracks) and [IGV](http://software.broadinstitute.org/software/igv/). This mitigates the need to load the much larger BAM files for data visualisation purposes which will be slower and result in memory issues. The bigWig format is also supported by various bioinformatics software for downstream processing such as meta-profile plotting. [bigBed](https://genome.ucsc.edu/goldenPath/help/bigBed.html) are more useful for displaying distribution of reads across exon intervals as is typically observed for RNA-seq data. Therefore, these files will only be generated if `--protocol directRNA` or `--protocol cDNA` are defined. @@ -112,7 +112,7 @@ The creation of these files can be bypassed by setting the parameters `--skip_bi * `/bigwig/` Per-sample `*.bigWig` files. -* `/bigbed/` +* `/bigbed/` Per-sample `*.bigBed` files. ## MultiQC @@ -120,14 +120,15 @@ The creation of these files can be bypassed by setting the parameters `--skip_bi *Documentation*: [MultiQC](https://multiqc.info/docs/) -*Description*: +*Description*: *MultiQC* is a visualisation tool that generates a single HTML report summarising all samples in your project. Most of the pipeline QC results are visualised in the report and further statistics are available within the report data directory. Results generated by *MultiQC* for this pipeline collate QC from *FastQC*, *samtools flagstat*, *samtools idxstats* and *samtools stats*. The pipeline has special steps which also allow the software versions to be reported in the *MultiQC* output for future traceability. For more information about how to use *MultiQC* reports, see . -*Output directories*: +*Output directories*: + * `multiqc/` * `multiqc_report.html` - a standalone HTML file that can be viewed in your web browser. * `multiqc_data/` - directory containing parsed statistics from the different tools used in the pipeline. From f943d324a90c2059f8c6874447684386cb4fd5ca Mon Sep 17 00:00:00 2001 From: drpatelh Date: Fri, 14 Feb 2020 15:45:04 +0000 Subject: [PATCH 21/22] Update docs --- docs/output.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/docs/output.md b/docs/output.md index ad86751f..86ad66c4 100644 --- a/docs/output.md +++ b/docs/output.md @@ -117,7 +117,7 @@ The creation of these files can be bypassed by setting the parameters `--skip_bi ## MultiQC -*Documentation*: +*Documentation*: [MultiQC](https://multiqc.info/docs/) *Description*: From 6e1e783bb31fa824092b691b77472b48654a3904 Mon Sep 17 00:00:00 2001 From: drpatelh Date: Fri, 14 Feb 2020 15:48:42 +0000 Subject: [PATCH 22/22] Change nanoplot image --- docs/images/nanoplot_readlengthquality.png | Bin 46836 -> 61857 bytes 1 file changed, 0 insertions(+), 0 deletions(-) diff --git a/docs/images/nanoplot_readlengthquality.png b/docs/images/nanoplot_readlengthquality.png index 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