Bowtie2 (2.5.1) reported very different alignment rates with and without --no-mixed --no-discordant

Ren-Meng commented 1 year ago

without --no-mixed --no-discordant

bowtie2 -p 20 -x ~/hg38.bowtie2.index -1 R1.fastq.gz -2 R2.fastq.gz --local --very-sensitive-local -S rep1.sam

39246458 reads; of these: 39246458 (100.00%) were paired; of these: 733800 (1.87%) aligned concordantly 0 times 23537958 (59.97%) aligned concordantly exactly 1 time 14974700 (38.16%) aligned concordantly >1 times

733800 pairs aligned concordantly 0 times; of these:
  31381 (4.28%) aligned discordantly 1 time
----
702419 pairs aligned 0 times concordantly or discordantly; of these:
  1404838 mates make up the pairs; of these:
    995116 (70.83%) aligned 0 times
    213569 (15.20%) aligned exactly 1 time
    196153 (13.96%) aligned >1 times

98.73% overall alignment rate

with --no-mixed --no-discordant

bowtie2 -p 20 -x ~/hg38.bowtie2.index -1 R1.fastq.gz -2 R2.fastq.gz --local --very-sensitive-local --no-mixed --no-discordant -S rep1.sam

39246458 reads; of these: 39246458 (100.00%) were paired; of these: 733800 (1.87%) aligned concordantly 0 times 30738446 (78.32%) aligned concordantly exactly 1 time 7774212 (19.81%) aligned concordantly >1 times 98.13% overall alignment rate

Why do --no-mixed and --no-discordant options affect concordant alignment rates?

ch4rr0 commented 1 year ago

Hello,

Thank you for reporting this issue. Would it be possible to share a sample of your reads that I can use to recreate this issue?

Ren-Meng commented 1 year ago

Thanks for your prompt reply~

Samples ENCFF763WUN and ENCFF658IDE (ENCSR769FOC) from ENCODE were used to perform this analysis.

And I also used Bowtie2 (2.4.5) to align reads from this paired-end sample to reference hg38. The concordant alignment rate with and without --no-mixed --no-discordant is the same.

bowtie2 (2.4.5) without --no-mixed --no-discordant

39246458 reads; of these: 39246458 (100.00%) were paired; of these: 733800 (1.87%) aligned concordantly 0 times 30792071 (78.46%) aligned concordantly exactly 1 time 7720587 (19.67%) aligned concordantly >1 times

733800 pairs aligned concordantly 0 times; of these:
  31381 (4.28%) aligned discordantly 1 time
----
702419 pairs aligned 0 times concordantly or discordantly; of these:
  1404838 mates make up the pairs; of these:
    995116 (70.83%) aligned 0 times
    213569 (15.20%) aligned exactly 1 time
    196153 (13.96%) aligned >1 times

98.73% overall alignment rate

bowtie2 (2.4.5) with --no-mixed --no-discordant

39246458 reads; of these: 39246458 (100.00%) were paired; of these: 733800 (1.87%) aligned concordantly 0 times 30792071 (78.46%) aligned concordantly exactly 1 time 7720587 (19.67%) aligned concordantly >1 times 98.13% overall alignment rate

ch4rr0 commented 1 year ago

Hello,

Thank you all the information provided. It was helpful in recreating the issue and finding the underlying cause. We are currently working on a fix. Thank you for your patience.

BenLangmead / bowtie2