Problem with alignment

[[email protected]]

Hello everyone!

We have received from BGI china a few samples we sent for exome sequence + Basic analysis. We got the fastQ files and BAM files. I have made my own BAM files using BWA and Picard tools using the following commends:

BWA:
bwa mem human_g1k_v37.fasta file1.fq.gz file2.fq.gz file3.fq.gz file4.fq.gz > result.sam

PICARD:
java -jar SortSam.jar I=result.sam O=result.bam SORT_ORDER=coordinate

The size of the files that I got (2Gb) , was about a half from the BAM files BGI (4.2 Gb) sent me.
Here are the samtools flagstat results for both files:

my BAM file:

[gen-biorep@gen-biorep-3 VA6]$ samtools flagstat result.bam
28842836 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 duplicates
28824861 + 0 mapped (99.94%:-nan%)
28842836 + 0 paired in sequencing
14423043 + 0 read1
14419793 + 0 read2
28593748 + 0 properly paired (99.14%:-nan%)
28814004 + 0 with itself and mate mapped
10857 + 0 singletons (0.04%:-nan%)
165631 + 0 with mate mapped to a different chr
131466 + 0 with mate mapped to a different chr (mapQ>=5)



BGI BAM File:

[gen-biorep@gen-biorep-3 result_alignment]$ samtools flagstat VA6.rmdup.bam
57668392 + 0 in total (QC-passed reads + QC-failed reads)
3039053 + 0 duplicates
57369744 + 0 mapped (99.48%:-nan%)
57668392 + 0 paired in sequencing
28834196 + 0 read1
28834196 + 0 read2
56991680 + 0 properly paired (98.83%:-nan%)
57273460 + 0 with itself and mate mapped
96284 + 0 singletons (0.17%:-nan%)
198072 + 0 with mate mapped to a different chr
181774 + 0 with mate mapped to a different chr (mapQ>=5

why does half of my reads disappear? How can I solve this problem?

Thank you,

Omri

[dpryan]

Unless you mislabeled the flagstat output, it looks like your results contain about twice as many alignments as those from BGI, though that could be due to yours containing multiple copies of multimappers while BGI's doesn't. In general, you'd need to look at how they aligned things and that will likely tell you why there's a difference.

[dpryan]

BTW, please don't cross post on both here and biostars, it creates duplicate work from the community.

[maubp]

Does either set of BAM file include unmapped read pairs? 'samtools idxstats example.bam' might help answer this.

P.S. Duplicate question was https://www.biostars.org/p/108537/

[[email protected]]

Sorry for the duplicate, won't happen again..

I'll try to remove the question from BioStars

BTW, please don't cross post on both here and biostars, it creates duplicate work from the community.

[[email protected]]

thank you. I have mislabled the flagstat ouputs, I have change it. BGI have more reads.

Unless you mislabeled the flagstat output, it looks like your results contain about twice as many alignments as those from BGI, though that could be due to yours containing multiple copies of multimappers while BGI's doesn't. In general, you'd need to look at how they aligned things and that will likely tell you why there's a difference.

[swbarnes2]

The .bam from bgi reports the exact same number of read 1 reads as read 2 reads. Yours does not. Are you sure that giving it 4 fastq files at once is the proper way to use bwa mem? I wonder if it is just ignoring those last two files. Maybe you could spot-check to see if read names from those fastqs are in your .bam

[[email protected]]

problem solved

The .bam from bgi reports the exact same number of read 1 reads as read 2 reads. Yours does not. Are you sure that giving it 4 fastq files at once is the proper way to use bwa mem? I wonder if it is just ignoring those last two files. Maybe you could spot-check to see if read names from those fastqs are in your .bam

Well i merged the 4 fastQ into one file and it did the trick. It's appear that BWA-MEM ignored from the last 2 files as you said. I got the right size of BAMs after that . Thank you swbarnes2 very much for your help!

[swbarnes2]

Well, don't merge them into 1 file, that's just throwing away real paired information.

You want all of the read 1 data in one fastq, and all the read 2 data in a second. Just make sure that the nth entry in the first fastq has the same read name as the nth read in file 2.