Good evening,

I'd like to compare the alignment quality of hisat2, bowtie2 and bwa for my files.

• The first 2 packages output the percentage of reads aligned concordantly exactly 1 time,
• bwa does not, because does not output alignment summary.

The samtools flagstat report is not enough, because it outputs only the general level of alignment, and I want to compare the other (percentage of reads aligned concordantly exactly 1 time).

I could filter the sam file by these reads, but I'm not sure I know how to do it correctly. I learned how to filter the sam file after hisat2 with the command samtools view -hf 0x2 -q 3,I have not learned how to filter the sam file after bowtie2 - none of the solutions I found worked, BUT in these cases I can at least check myself. In the case of bwa, I can't check myself due to the lack of alignment summary.

Could you please advise how to get a fraction of reads aligned concordantly exactly 1 time, and extract them from the sam file after bwa (version 0.7.17)?

EDIT:

I tried samtools view -f 0x2 | grep -v -e 'XA:Z:' -e 'SA:Z:' (based on the combination of the previous link and this), but obtained unpaired reads in the final file. XA:Z is it is put down by bwa if there are alternative alignments. But in some cases XA:Z exist in only one string of pair, for example:

ERR3316120.48768420     99      NODE_5_length_137336913_cov_22.55       93837250        60      24S127M =       93837315        216     GTTGAACCATGGCACCCC
TTGTTAAGGCTACCTTTTGCATGCCCAGAGATGCAAGCACCAAGTTCTGCTATCAATTTACATTGTGACAGTTTGCAGATGACTTCTGCACGCACCACGTGTCCTGGACACATGGAATTCTCTTTTGCTGGCC   AA<A<FJJJF7-AFAJAJ
JJJJJJFFFJJFFJ7JJJFFFJJJF<JFFFJJAFF<FJJJJ<JFFFJJJFJFJJ--JAAJJJJFJ-7FF-AJJJJFJA-F<FFFJJJFJFJFAFFJJJJJFJFJFFAAFJJFJJFA<-FFJFF-FAAJJAFFA   NM:i:0  MD:Z:127 M
C:Z:151M        AS:i:127        XS:i:92
ERR3316120.48768420     147     NODE_5_length_137336913_cov_22.55       93837315        60      151M    =       93837250        -216    TTTGCAGATGACTTCTGC
ACGCACCACGTGTCCTGGACACATGGAATTCTCTTTTGCTGGCCTGTATCAATGTTTTGGATCTTTTTCTACGTTCAGGCTATGTAAACATTCATTTCTAACGACAAATGAATTCCTTGAGTTACATATTTAA   JFFAFAFJJJJAJFJA7<
A<FAAJAJJJJF7<<FFJF<FFJFFJ<FA-FA-FF7FAFJFF-JJJJJAAJJJJJ<JFJFJJFA<FF<7FF-<AF7AFA-JJJJJJA7FJJJJJJFF<JJJFAF-J-AJJJJFAAF<JFJFJFFFFJAFAAA-   NM:i:0  MD:Z:151 M
C:Z:24S127M     AS:i:151        XS:i:123        XA:Z:NODE_5_length_137336913_cov_22.55,-93864831,151M,6;

So, in the final file I have only the first string. Should I remove such alignments manually, using smth like this?

Thank you in advance!

Best regards, Poecile