Hi,

I am trying to run diffHic on a single chromosome. I extracted the chromosome 1 alignment data and used the bam file as an input for diffHic. However, at the preparePairs() stage, none of the reads appear as paired.

This is the output from diffHic:

diagnostics <- preparePairs("chr1.bam", hs.param, file="chr1.h5", dedup=TRUE, minq=10)
> diagnostics
$pairs
   total   marked filtered   mapped 
       1        0        0        1 
$same.id
   dangling self.circle 
          0           0 
$singles
[1] 20248727
$chimeras
  total  mapped   multi invalid 
      0       0       0       0 

When I checked the bam file with samtools flagstat, I get:

samtools flagstat chr1.bam 
20248729 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
20248729 + 0 mapped (100.00% : N/A)
20248729 + 0 paired in sequencing
10125154 + 0 read1
10123575 + 0 read2
20248729 + 0 properly paired (100.00% : N/A)
20248729 + 0 with itself and mate mapped
0 + 0 singletons (0.00% : N/A)
1240765 + 0 with mate mapped to a different chr
1240765 + 0 with mate mapped to a different chr (mapQ>=5)

So the reads are paired but preparePairs() does not seem to recognise this. Any suggestions?

Thank you very much!

> sessionInfo()
R version 3.2.3 (2015-12-10)
Platform: x86_64-apple-darwin13.4.0 (64-bit)
Running under: OS X 10.10.5 (Yosemite)
locale:
[1] C/UTF-8/C/C/C/C
attached base packages:
[1] stats4    parallel  stats     graphics  grDevices utils     datasets 
[8] methods   base     
other attached packages:
[1] diffHic_1.2.2        GenomicRanges_1.22.4 GenomeInfoDb_1.6.3  
[4] IRanges_2.4.7        S4Vectors_0.8.11     BiocGenerics_0.16.1 
loaded via a namespace (and not attached):
 [1] AnnotationDbi_1.32.3       XVector_0.10.0            
 [3] edgeR_3.12.0               zlibbioc_1.16.0           
 [5] GenomicAlignments_1.6.3    BiocParallel_1.4.3        
 [7] lattice_0.20-33            BSgenome_1.38.0           
 [9] tools_3.2.3                grid_3.2.3                
[11] SummarizedExperiment_1.0.2 rhdf5_2.14.0              
[13] Biobase_2.30.0             csaw_1.4.1                
[15] DBI_0.3.1                  lambda.r_1.1.7            
[17] futile.logger_1.4.1        rtracklayer_1.30.2        
[19] futile.options_1.0.0       bitops_1.0-6              
[21] biomaRt_2.26.1             RCurl_1.95-4.7            
[23] RSQLite_1.0.0              limma_3.26.8              
[25] GenomicFeatures_1.22.13    Biostrings_2.38.4         
[27] Rsamtools_1.22.0           locfit_1.5-9.1            
[29] XML_3.98-1.3 

 

Check that the BAM file is name sorted. Also, preparePairs identifies read pairs based on having identical alignment names. If that's not the case (e.g., there's /1 or /2 appended to the name) the function will fail.