Low pairing rate

[jeremymsimon]

Hi-
I have some H3K27ac HiChIP data that I'm processing and I seem to be getting a low proper pairing rate despite relaxing to --very-sensitive --end-to-end --reorder in the local and global bowtie2 options, see below for stats from the *.pairstat files in the bwt2 output directory.

Do you have any suggestions on how to improve my paired alignment rate or otherwise rescue more of the singleton reads here?

The setup, as far as I'm aware, is with MNase rather than any restriction enzyme, so my config is rather vanilla already. After running HiC-Pro, I am running FitHiChIP and although I do get some significant interactions, it isn't as many as I would have anticipated.

Note too that running macs2 on my two BAM files here does give me 37414 peaks, which is about right for this histone modification.

Thanks!

[Rep 1]
Total_pairs_processed	104940393	100.0
Unmapped_pairs	14820725	14.123
Low_qual_pairs	15802354	15.058
Unique_paired_alignments	22710085	21.641
Multiple_pairs_alignments	0	0.0
Pairs_with_singleton	51607229	49.178
Low_qual_singleton	0	0.0
Unique_singleton_alignments	0	0.0
Multiple_singleton_alignments	0	0.0
Reported_pairs	22710085	21.641

[Rep 2]
Total_pairs_processed	123039150	100.0
Unmapped_pairs	16294921	13.244
Low_qual_pairs	19153987	15.567
Unique_paired_alignments	26760375	21.749
Multiple_pairs_alignments	0	0.0
Pairs_with_singleton	60829867	49.439
Low_qual_singleton	0	0.0
Unique_singleton_alignments	0	0.0
Multiple_singleton_alignments	0	0.0
Reported_pairs	26760375	21.749

Relevant snippets of config:

#######################################################################
## Alignment options
#######################################################################

MIN_MAPQ = 10

BOWTIE2_IDX_PATH = /path/to/bowtie2_indexes/bowtie_v251/hg38/
BOWTIE2_GLOBAL_OPTIONS = --very-sensitive --end-to-end --reorder
BOWTIE2_LOCAL_OPTIONS =  --very-sensitive --end-to-end --reorder

[...]

#######################################################################
## Allele specific analysis
#######################################################################

ALLELE_SPECIFIC_SNP = 

#######################################################################
## Capture Hi-C analysis
#######################################################################

CAPTURE_TARGET =
REPORT_CAPTURE_REPORTER = 1

#######################################################################
## Digestion Hi-C
#######################################################################

GENOME_FRAGMENT = 
LIGATION_SITE = 
MIN_FRAG_SIZE = 
MAX_FRAG_SIZE =
MIN_INSERT_SIZE =
MAX_INSERT_SIZE =

#######################################################################
## Hi-C processing
#######################################################################

MIN_CIS_DIST =
GET_ALL_INTERACTION_CLASSES = 1
GET_PROCESS_SAM = 0
RM_SINGLETON = 1
RM_MULTI = 1
RM_DUP = 1

#######################################################################
## Contact Maps
#######################################################################

BIN_SIZE = 5000 10000 20000 40000 150000 500000 1000000 2000000
MATRIX_FORMAT = upper

#######################################################################
## Normalization
#######################################################################
MAX_ITER = 100
FILTER_LOW_COUNT_PERC = 0.02
FILTER_HIGH_COUNT_PERC = 0
EPS = 0.1