ERROR SAMTOOLS GENERATING TRUNCATED FILE: SEQ and QUAL are of different length

Question

[meilinfer]

I would like to convert a bowtie2 generated .sam file to .bam with samtools (v1.9) but I get truncated files with SEQ and QUAL of different lenghts. The error line SEQ and QUAL's are indeed of different lenght but I don't know how to fix this.Why am I getting this error?

$ samtools view -b hiPSC1.sam > hiPSC1.bam

[E::sam_parse1] SEQ and QUAL are of different length

[W::sam_read1] Parse error at line 8272508

[main_samview] truncated file

here's how my .sam file looks:

@SQ SN:chrY LN:57227415

SRR2097503.1.3  83  chrM    6842    1   51M =   6745    -148    TATCCCCACCGGCGTCAAAGTATTTAGCTGACTCGCCACACTCCACGGAAG >>?8/:AA6A@?0DB9DB<?B@A@<DDC?)1EEC=CA?A+D?DDD@;D??? AS:i:0  XS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:51 YS:i:0  YT:Z:CP

and here is the line giving me the error:

$ sed -n 8272508p hiPSC1.sam

SRR2097503.1.4850276    99  chrM    14650   34  51M =   14804   205 CAACAGAAACAAAGCATACATCATTATTCTCGCACGGACTACAACCACGAC CCCFFFFFHHHHHIJJJJJJJJJJJJJJJJJJJJJIGGDJIJJIGJHHHHHFFFFFCCC AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:51 YS:i:0  YT:Z:C

[GenoMax]

Please go back and check your original data file to see if the problem exists there as well.

[ATpoint]

The SAM file is corrupted. Maybe an issue during alignment, amybe out-of-memory at some point. I would realign everything from scratch. If it is only this one read then you can also remove it and see if that fixes it. I would still realign:

bowtie2 (...option) | samtools view -o out.bam

which will produce a BAM file right away without intermediate sam files. This is called a Unix pipe.

[meilinfer]

Thanks AT point I used bowtie2 to align and use a prebuilt index I downloaded from Bowtie's webpage. I'll try realigning again and piping samtools

bowtie2 --very-sensitive --no-discordant --no-mixed -X 2000 -x grch38_1kgmaj -1 hiPSC1_1P.fastq -2 hiPSC1_2P.fastq -p 4 > hiPSC1.sam

[GenoMax]

You could use a pipe as suggested by @ATPoint or use the correct option to create the SAM file instead of the redirect you used.

bowtie2 --very-sensitive --no-discordant --no-mixed -X 2000 -x grch38_1kgmaj -1 hiPSC1_1P.fastq -2 hiPSC1_2P.fastq -p 4 -S hiPSC1.sam

[meilinfer]

I did not use the -S!! Thanks I'll give it a go and align again. Thanks.

Answer 1

[ATpoint]

Your command does not contain a pipe. That would be:

bowtie2 --very-sensitive --no-discordant --no-mixed -X 2000 -x grch38_1kgmaj -1 hiPSC1_1P.fastq -2 hiPSC1_2P.fastq -p 4 | samtools view -o hiPSC1.bam

[meilinfer]

It worked!! I checked the file with:

samtools quickcheck -v

and it was okay and was even able to sort the file.Thanks!

[ATpoint]

Cool, glad to hear.

Answer 2

[Pierre Lindenbaum]

It's a problem with your SAM, not with samtools. It looks like a problem in your upstream process and you should not trust this data.

You can always filter out the bad lines with awk....

awk -F '\t' '$0 ~ /^@/ || length($10)==length($11)' input.sam