Problems sam > bam conversion

Question

[Pinca]

Hi all,

I am currently analyzing a pop-seq dataset and encounter some problems when trying to convert my .sam file into a .bam file.

Here is what i did so far:

1) I indexed the genome using Bowtie2 v. 2.4.2

bowtie2-build GCF_000001215.4_Release_6_plus_ISO1_MT_genomic.fna philip_indexed_genome

2) I then mapped the reads using Bowtie2 v. 2.4.2

bowtie2 -x philip_indexed_genome -1 b2_HCHKKDSXY_L1_1.fq -2 b2_HCHKKDSXY_L1_2.fq -S b2_bowtie2_to_indexed_genome.sam

When looking at the output, the mapping seems to have been successful:

302546831 reads; of these:

302546831 (100.00%) were paired; of these:

31467015 (10.40%) aligned concordantly 0 times

217839984 (72.00%) aligned concordantly exactly 1 time

53239832 (17.60%) aligned concordantly >1 times

---

31467015 pairs aligned concordantly 0 times; of these:

10785696 (34.28%) aligned discordantly 1 time

---

20681319 pairs aligned 0 times concordantly or discordantly; of these:

41362638 mates make up the pairs; of these:

26239879 (63.44%) aligned 0 times

7729764 (18.69%) aligned exactly 1 time

7392995 (17.87%) aligned >1 times

95.66% overall alignment rate

3) However, when trying to convert the resulting .sam file, into a .bam file using (I used samtools 1.9)

samtools view -Sb b2_bowtie2_to_indexed_genome.sam > b2_bowtie2_to_indexed_genome.bam

I get the following message:

[W::sam_read1] Parse error at line 545655373

[main_samview] truncated file.

I tried to look at that line using

sed -n 545655372p b2_bowtie2_to_indexed_genome.sam > output_from_sam2

but the output file is empty. Besides, I counted the number of rows in the sam file using

wc -l b2_bowtie2_to_indexed_genome.sam

and got

198853022

which is less than the line flagged as problematic.

Does anybody have an idea of what's going on? Thanks a lot Philip

Answer 1

[ATpoint]

Something went wrong during alignment. Before digging deeper simply repeat alignment and see how it goes. You can produce bam right away using pipes, there is no reason to keep the sam file rather than the bam. Be sure to use recent samtools versions.

bowtie2 (options...) -1 b2_HCHKKDSXY_L1_1.fq -2 b2_HCHKKDSXY_L1_2.fq | samtools view -o out.bam

[Pinca]

This solved the problem. Thanks!

[Pinca]

Thanks for the reply. Ill try that!