A while ago I have done a training on NextGen analysis. Recently I am trying to do the same analysis for the same Genome sequence on a different machine but now when i compare the flagstat results from samtools of the merged files they are different.
Methods: I used BWA for the alignment -q 20 to get .sai files > then convert these to .sam files using bwa sampe > later i convert these to .bam files using samtools /samtools view -bS | /samtools sort -. So my question is what went wrong ? is it normal that i get different results each time i do an alignment ? if so why I am getting a less properly paired in my recent analysis.
(Recent analysis) of the final merged .bam files samtools flagstat:
253230842 + 0 in total (QC-passed reads + QC-failed reads) 493185 + 0 duplicates 236903690 + 0 mapped (93.55%:nan%) 253230842 + 0 paired in sequencing 126615421 + 0 read1 126615421 + 0 read2 232042648 + 0 properly paired (91.63%:nan%) 234896807 + 0 with itself and mate mapped 2006883 + 0 singletons (0.79%:nan%) 1946513 + 0 with mate mapped to a different chr 778386 + 0 with mate mapped to a different chr (mapQ>=5)
While doing the training samtools results of the same genome sequence
335964388 + 0 in total (QC-passed reads + QC-failed reads) 0 + 0 duplicates 313032349 + 0 mapped (93.17%:nan%) 335964388 + 0 paired in sequencing 167982194 + 0 read1 167982194 + 0 read2 305304034 + 0 properly paired (90.87%:nan%) 309497738 + 0 with itself and mate mapped 3534611 + 0 singletons (1.05%:nan%) 2984962 + 0 with mate mapped to a different chr 1131183 + 0 with mate mapped to a different chr (mapQ>=5)
Thanks a lot in advance