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GATK flagstats show 0.0 % properly paired reads
MUHAMMADSOHAILRAZA
Beijing Institute of Genomics, CASMember ✭✭
Hi everyone,
I used GATK Flagstats walker. I am getting 0.0% properly paired reads in the BAM file. I sorted my BAM file and marked the duplicates as well. is there any BAM FLAG is missing in my file or mapping results are bad quality. Note that my average mapping quality (MAPQ) is more than 30.. and average read legth is 150 bp. i used bwa-mem for mapping to reference genome. and processed my data with picard-tools before using in GATK.
can you please help help me tracking this issue?
my GATK arguments were:
java -Xmx15g -jar /GTK/GenomeAnalysisTK.jar -T FlagStat \
-log Flagstat.log \
-R /Ref/human_g1k_v37.fasta \
-I /CHG000691/Dedup_Sort_CHG000691_PE.bam \
-o /CHG000691/Dedup_Sort_CHG000691_flagstat.txt
Output:
606634523 in total
0 QC failure
24671783 duplicates
599254096 mapped (98.78%)
606634523 paired in sequencing
303086535 read1
303547988 read2
0 properly paired (0.00%)
593494816 with itself and mate mapped
5759280 singletons (0.95%)
28638158 with mate mapped to a different chr
9047446 with mate mapped to a different chr (mapQ>=5)
Thank you very much!
Answers
The properly paired flag is set by the aligner, GATK is merely reporting what's stored in the file. You should look into the documentation for your aligner to see what it considers to be "properly paired" (and keep in mind, some aligners may not ever set that flag)
I am facing a similar issue with a particular sample from a group of samples that I am analyzing. All other samples have over 95% properly paired reads but in one of my sample the % mapped in 99.2% and the %properly mapped is 0.0%.
Could someone please explain to me what could be going on with my samples ? Thanks a lot
@aditisk, we can't help you with that. See @pdexheimer's answer above.
@aditisk @Geraldine_VdAuwera
I found that While mapping reads to reference sequence in BWA, you need to carefully set the parameters, like for example in the BWA command:
bwa mem -t 5 -R '@RG\tID:02AL1\tSM:HMN15-3\tLB:hs\tPL:illumina' -P -M human_g1k_v37.fasta /RAWReads/S17_02A_CHG000693_L001_R1.fastq.gz /RAWReads/S17_02A_CHG000693_L001_R2.fastq.gz > /Results/CHG000693_PE_02AL1.sam
In the above command by using the parameter -P leads to "0.0% properly paired reads" output. Because:
-P: paired-end mode, perform SW to rescue missing hits only but do not try to find hits that fit a proper pair.
I hope this will help.. This is not GATK issue.
Hmm, interesting -- I was not aware of that BWA setting. Thank you for pointing this out!
@MUHAMMADSOHAILRAZA Thanks for pointing that out. I will be more careful with my BWA commands from the next time.
@aditisk
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