BWA-mem alignment is not giving desired output

Question

[micro32uvas]

Hello, I have clean and trimmed fastq files with an average of 150nt per read. I have tried BWA aln and also BWA mem for the alignment with my reference genome.

The alignment take around 2 days to process but ends u with 92kb file each time. I have changed algorithm from aln to mem, tried default parameters, tried clean fq and trimmed fq seperately. but same result all the time. Here are the commands that i used:

bwa index -a is ref.fna bwa aln ref.fna 1.fastq >1.aln bwa aln ref.fna 2.fastq >2.aln both files yileded alignment but when i used sampe: bwa sampe -P ref.fna 1.aln 2.aln 1.fastq 2.fastq > sample.sam.all it yielded 92kb

Later i tried BWA mem bwa mem -t 24 -M ref.fna trimmed-files1/1P.fastq trimmed-files1/2P.fastq >sample.sam 2> mem-pe.log also yielded 92kb file.

I cannot understand the reason. I checked the quality and it is all good.

Your help is appreciated. Qurat

[Tm]

Please check the BWA log messages and see whether BWA was completed normally or with error? This may help you solve your query.

Answer 1

[mittu1602]

Use the following commands with following parameters:

bwa mem ucsc.hg19.fasta 1.fastq 2.fastq -M   > test.alnpe.sam
samtools view -Sb test.alnpe.sam >test_bam.alnpe.bam
samtools "sort" -@ 24 test_bam.alnpe.bam >test_bam.alnpe.sort.bam

[micro32uvas]

I am indexing the genome again with bwa. However, the command that you indicated; isnt it the same as i mentioned above?

bwa mem -t 24 -M ref.fna trimmed-files1/1P.fastq trimmed-files1/2P.fastq >sample.sam 2> mem-pe.log

Kindly elaborate it a little. I cannot get the desired output with the same command

[mittu1602]

The commands could be quoted by "code sample" separately so that it is easy for the user to read, also yes the commands are same but I would suggest you to try once with reference file as ".fasta/.fa" rather ".fna" because BWA MEM is not able to read the reference file and therefore not able to process.

[micro32uvas]

now that i am trying with .fa indexed file. Here is the next error, that needs to be resolved:

[E::bwa_set_rg] the read group line is not started with @RG

and here is my read group:

@A00234:29:H5LTLDMXX:2:2488:31855:37059 1:N:0:GCTGGTTA

I read all the "passing a variable to the read group"but i couldnt get it right.

Your hep is appreciated.

[GenoMax]

What does mem-pe.log contain?

[micro32uvas]

It just includes the intermediate processes.