Question: BWA-mem alignment is not giving desired output
0
gravatar for micro32uvas
12 months ago by
micro32uvas10
Pakistan
micro32uvas10 wrote:

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

wgs alignment bwa-mem • 686 views
ADD COMMENTlink modified 12 months ago by mittu1602150 • written 12 months ago by micro32uvas10

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

ADD REPLYlink written 12 months ago by toralmanvar720
1
gravatar for mittu1602
12 months ago by
mittu1602150
India
mittu1602150 wrote:

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
ADD COMMENTlink written 12 months ago by mittu1602150

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

ADD REPLYlink modified 12 months ago by genomax59k • written 12 months ago by micro32uvas10

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.

ADD REPLYlink written 12 months ago by mittu1602150

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.

ADD REPLYlink written 12 months ago by micro32uvas10

What does mem-pe.log contain?

ADD REPLYlink written 12 months ago by genomax59k

It just includes the intermediate processes.

ADD REPLYlink written 12 months ago by micro32uvas10
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