BWA MEM output has read1 sequence written into read2 record — input FASTQs are correctly paired
I am a beginner at using BWA. I've encountered a strange problem with BWA MEM and would appreciate any insight.In the SAM output, the sequence (SEQ field) of read1 has been written into the record of read2, and vice versa.
ERR12731759.15015565 99 2 23621249 0 29S20M59S = 23621249 20 **TAAGAAAGAACAGTATGATCTTTCTGTAGATAATGAAGATTATAATATTCAGTTGAGTAGTACTGCAGATTATTCTCCTGAAGATATCTTCATTGGAAATGAAACTCA** **AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEAEEE/EEEEEEEEAEEEEEEEEEEEEEEEEEEAE** NM:i:0 MD:Z:20 MC:Z:29S20M59S AS:i:20 XS:i:19 XA:Z:chr10,+127215122,37S19M52S,0;15,+102716716,39S19M50S,0;6,-82318477,58S19M31S,0;
ERR12731759.15015565 147 2 23621249 0 29S20M59S = 23621249 -20 **TAAGAAAGAACAGTATGATCTTTCTGTAGATAATGAAGATTATAATATTCAGTTGAGTAGTACTGCAGATTATTCTCCTGAAGATATCTTCATTGGAAATGAAACTCA** **EEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEAEEEEEEEEAEEEEEEEEEAEEAEEEEEEEEEEEEEEEEEEEEEE/EEEEEEEEEEEEEEEEEEEEAEEEAAAAA** NM:i:0 MD:Z:20 MC:Z:29S20M59S AS:i:20 XS:i:19 XA:Z:6,+82318477,58S19M31S,0;15,-102716716,39S19M50S,0;chr10,-127215122,37S19M52S,0;
This issue occurs with the last pair of reads. Their quality scores are correct, but the read sequences are wrong. I've checked the input files and found no problems.
ERR12731759_1_paired.fastq.gz tail
@ERR12731759.15015565 NB501204:173:HM2MMBGXK:4:23612:9993:4160 length=108
**TAAGAAAGAACAGTATGATCTTTCTGTAGATAATGAAGATTATAATATTCAGTTGAGTAGTACTGCAGATTATTCTCCTGAAGATATCTTCATTGGAAATGAAACTCA**
+ERR12731759.15015565 NB501204:173:HM2MMBGXK:4:23612:9993:4160 length=108
**AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEAEEE/EEEEEEEEAEEEEEEEEEEEEEEEEEEAE**
(bwa) ps@ps:/mnt/sda/ohb_file/fa/ERR/trim$ zcat ERR12731759_2_paired.fastq.gz |tail
+ERR12731759.15015563 NB501204:173:HM2MMBGXK:4:23612:9988:14129 length=148
AAAAAAEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE6EEEEEEE<EEEEEEEEEEEEEEEEEAEEA/EAEAAA/EEEEEEEEEAEA<A<EEAEEAA<AAAEAAA/6AAA/<<<A///6
@ERR12731759.15015564 NB501204:173:HM2MMBGXK:4:23612:9989:4742 length=148
GGCACGAACACGGGAATAGGCATTTTGCGTATCTGTTACAAACGGAACAAACAAAATCTGCATGCCTTGATCGGCCATCAATCGCGAAAGTTCGGGAAACTCCACATCGTAGCAAATCAATGCACCGATTCTGGCACAATCGGTGTCG
+ERR12731759.15015564 NB501204:173:HM2MMBGXK:4:23612:9989:4742 length=148
AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE/EEEEEEEEEEEEEEEEEE<EEEEAEEEAEEEEAEEEEA<EEAEEAEEEEEEEA<<EEAEEAAEEEE<EEEEEEEEAEEEA/A<<</<AEEAE<AAEEEEEAE<AAA
@ERR12731759.15015565 NB501204:173:HM2MMBGXK:4:23612:9993:4160 length=108
**TGAGTTTCATTTCCAATGAAGATATCTTCAGGAGAATAATCTGCAGTACTACTCAACTGAATATTATAATCTTCATTATCTACAGAAAGATCATACTGTTCTTTCTTA**
+ERR12731759.15015565 NB501204:173:HM2MMBGXK:4:23612:9993:4160 length=108
**AAAAAEEEAEEEEEEEEEEEEEEEEEEEE/EEEEEEEEEEEEEEEEEEEEEEAEEAEEEEEEEEEAEEEEEEEEAEEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEE**
[main] Version: 0.7.17-r1188
[main] CMD: bwa mem -t 64 -M /mnt/sda/ohb_file/fa/omg/host/host.fa ERR12731759_1_paired.fastq.gz ERR12731759_2_paired.fastq.gz
[main] Real time: 416.662 sec; CPU: 14414.652 sec
This is the command I used, and there were no error messages during the entire run.”
Why does BWA output read1's sequence twice, but the quality values are from read2 and read1? I have repeated this twice with the same outcome.
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I found this issue in other files.I also upgraded bwa to the latest version, but the output still has this issue. Is this normal?
what do you mean. I see the sequence 'TAAGAAAGAACAGTATGATCTTTCTGTAGATAATGAAGATTATAATATTCAGTTGAGTAGTACTGCAGATTATTCTCCTGAAGATATCTTCATTGGAAATGAAACTCA' in the first read (first in pair) of the first SAM alignement
unless I don't understand your question.
Thank you for your reply. I think I have already figured out where the problem was. I misunderstood the SAM file — this is actually the output of the reverse-complement sequence.I'm very sorry.