Attempting to generate a bam.bai file but the output is not readable

Question

[K.patel5]

Hi,

I am new a exome sequencing, and have tried to follow tutorials on the subject. I am stuck at the samtools index stage because the output files are in a non-human readable format and I believe I am making a misstep somewhere. Below I have my code and I have the head of the outputted .bam.bai file.

For simplicity we will assume all files are in the same folder.

#concatenate lanes
cat L001_R1_001.fastq.gz L002_R1_001.fastq.gz L003_R1_001.fastq.gz L004_R1_001.fastq.gz > subject_R1.fastq.gz  
cat L001_R2_001.fastq.gz L002_R2_001.fastq.gz L003_R2_001.fastq.gz L004_R2_001.fastq.gz > subject_R2.fastq.gz  

#index genome
bwa index -t 8 GCA_000001405.15_GRCh38_no_alt_analysis_set.fna GCA_000001405.15_GRCh38_no_alt

#create bam file. Sam file creation is piped to save space.
bwa mem -t 8 GCA_000001405.15_GRCh38_no_alt_analysis_set.fna subject_R1.fastq.gz   subject_R2.fastq.gz -M \
-R "@RG\tID:FlowCell.subject1\tSM:subject1\tPL:illumina\tLB:mito.subject1"   | \
samtools sort -O bam -o ${bamfolder}/subject1_bwa_output.bam 

#create bam.bai file
samtools index -b subject1_bwa_output.bam

#check the bam.bai file
samtools flagstat subject1_bwa_output.bam.bai > subject1_stat_bwa_output.txt 
samtools idxstats subject1_bwa_output.bam.bai > subject1_idxstat_bwa_output.txt

During the checking phase I am getting the following errors.

[E::hts_hopen] Failed to open file subject1_bwa_output.bam.bai
[E::hts_open_format] Failed to open file "subject1_bwa_output.bam.bai" : Exec format error
samtools flagstat: Cannot open input file "subject1_bwa_output.bam.bai": Exec format error
[E::hts_hopen] Failed to open file subject1_bwa_output.bam.bai
[E::hts_open_format] Failed to open file "subject1_bwa_output.bam.bai" : Exec format error
samtools idxstats: failed to open "subject1_bwa_output.bam.bai": Exec format error

Here is a screen-shot of the output from the bam.bai file. I feel like this is not correct.

Answer 1

[zorbax]

you should apply flagstat to the bam (previously indexed) file: samtools flagstat subject1_bwa_output.bam.

[K.patel5]

Thanks, that worked.

[ATpoint]

fyi, the bai file is an index, you do not ever need to interact with it directly. If a tool needs it it will look for it in the same folder as the main bam file automatically. It does not contain human-readable content that could be of interest.

[K.patel5]

This makes sense. I had assumed the bam.bai file is a summarized copy of the .bam file. Thank you!