Samtools issues with bai files

Question

[njmcdonald]

I have created a sam file using bowtie2. I need to convert it to a bam file, index it an do downstream variant calling. However, every attempt I've made to make an index file results in an an unusable file.

One approach:

[guest@centos6 wu2]$ samtools view -h wu_0.sorted.bam | head

@HD VN:1.0 SO:coordinate

@SQ SN:Chr1 LN:29923332

@SQ SN:Chr2 LN:19386101

@SQ SN:Chr3 LN:23042017

@SQ SN:Chr4 LN:18307997

@SQ SN:Chr5 LN:26567293

@SQ SN:chloroplast LN:154478

@SQ SN:mitochondria LN:366924

@PG ID:bowtie2 PN:bowtie2 VN:2.2.5 CL:"/usr/local/bin/bowtie2-align-s --wrapper basic-0 -x wu_0_A/wu_0_A wu_0_A_wgs.fastq -s wu_0_bt2.sam"

unknown:2:60:1525:1235/1 0 Chr1 396 42 50M * 0 0 TTACTCCTTTGTGGAAATGTTTGTTCTATCAATTTATCTTTTGTGGGAAA CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:50 YT:Z:UU

I then enter the command to index the file...

$ samtools index wu_0.sorted.bam

I confirm I have a file with the appropriate extension:

wu_0.sorted.bam.bai

I try to run mpileup command and the following errors result:

$ samtools mpileup -v -u -f wu_0.v7.fas wu_0.sorted.bam.bai > wu.vcf

[E::hts_hopen] fail to open file 'wu_0.sorted.bam.bai'

[E::hts_open] fail to open file 'wu_0.sorted.bam.bai'

[mpileup] failed to open wu_0.sorted.bam.bai: Success

Then I tried the following:

[guest@centos6 wu2]$ samtools view -bS wu_0_bt2.sam > wu_0.bam

[guest@centos6 wu2]$ samtools sort wu_0.bam wu.sorted

[guest@centos6 wu2]$ samtools index wu.sorted.bam

[guest@centos6 wu2]$ ls

wu_0_A  wu_0_A_wgs.fastq  wu_0.bam  wu_0_bt2.sam  wu_0_local_bt2.sam  wu_0.v7.fas  wu.sorted.bam  wu.sorted.bam.bai

[guest@centos6 wu2]$ samtools mpileup -v -u -f wu_0.v7.fas wu.sorted.bam.bai > wu.vcf

[fai_load] build FASTA index.

[E::hts_hopen] fail to open file 'wu.sorted.bam.bai'

[E::hts_open] fail to open file 'wu.sorted.bam.bai'

[mpileup] failed to open wu.sorted.bam.bai: No such file or directory

So what is going wrong?

[GenoMax]

Is the .bai file non-zero bytes (i.e. non empty) in both cases?

Answer 1

[Devon Ryan]

You meant to execute samtools mpileup -v -u -f wu_0.v7.fas wu_0.sorted.bam > wu.vcf. You don't specify the .bai files in commands; the files are loaded as needed.