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Hi everybody,
We are having an irritating problem with our BWA workflow which we have been working on for the last few days.
The symptoms of our problem are the following:
1) Our snpFiltering script that has worked with pileup files created after a maq workflow do not work with our new bwa-generated data. (While the indel filtering script under samtools works, the results are probably not reliable because of this and the other problems below.)
2) To go back to the previous step, the sorted and indexed .bam file that we used the pileup command on, cannot be viewed under tview pointing at a fault.
3) Taking one further step back, we have determined that integrating the .sam file generated from bwa back into the maq workflow did not create a meaningful output as well. So the fault should already be before this step.
We are using the latest version of Solexa raw sequence data format as our data is relatively recent. We did use the correct fastq conversion algorithm and confirm that the files are valid after a successful maq workflow.
The current workflow that we are using under bwa-samtools is the following as written as part of a shell script designed to work in the folder that contains the data.
bwa index -a bwtsw chr11.fa
bwa aln -e $i(3 to 8) chr11.fa sequence1.fastq > sequence1_indexed.sai
bwa aln -e $i(3 to 8) chr11.fa sequence2.fastq > sequence2_indexed.sai
bwa samse chr11.fa sequence1_indexed.sai sequence1.fastq > sequence1_aligned.sam
bwa samse chr11.fa sequence2_indexed.sai sequence1.fastq >sequence2_aligned.sam samtools faidx chr11.fa
samtools view -bt chr11.fa.fai -o sequence1_aligned.bam sequence1_aligned.sam
samtools view -bt chr11.fa.fai -o sequence2_aligned.bam sequence2_aligned.sam
samtools sort sequence1_aligned.bam sequence1_aligned_sorted
samtools sort sequence2_aligned.bam sequence2_aligned_sorted
samtools merge merged_sequence.bam sequence1_aligned_sorted.bam sequence2_aligned_sorted.bam
samtools sort merged_sequence.bam merged_sequence_sorted
samtools index merged_sequence_sorted.bam
samtools pileup -f chr11.fa -c NG8_s_merged_sequence_sorted.bam > final_alignment.pileup
A run without using data from both lanes produced a similar unusable output.
Any help would be appreciated.
Thanks for reading.
We are having an irritating problem with our BWA workflow which we have been working on for the last few days.
The symptoms of our problem are the following:
1) Our snpFiltering script that has worked with pileup files created after a maq workflow do not work with our new bwa-generated data. (While the indel filtering script under samtools works, the results are probably not reliable because of this and the other problems below.)
2) To go back to the previous step, the sorted and indexed .bam file that we used the pileup command on, cannot be viewed under tview pointing at a fault.
3) Taking one further step back, we have determined that integrating the .sam file generated from bwa back into the maq workflow did not create a meaningful output as well. So the fault should already be before this step.
We are using the latest version of Solexa raw sequence data format as our data is relatively recent. We did use the correct fastq conversion algorithm and confirm that the files are valid after a successful maq workflow.
The current workflow that we are using under bwa-samtools is the following as written as part of a shell script designed to work in the folder that contains the data.
bwa index -a bwtsw chr11.fa
bwa aln -e $i(3 to 8) chr11.fa sequence1.fastq > sequence1_indexed.sai
bwa aln -e $i(3 to 8) chr11.fa sequence2.fastq > sequence2_indexed.sai
bwa samse chr11.fa sequence1_indexed.sai sequence1.fastq > sequence1_aligned.sam
bwa samse chr11.fa sequence2_indexed.sai sequence1.fastq >sequence2_aligned.sam samtools faidx chr11.fa
samtools view -bt chr11.fa.fai -o sequence1_aligned.bam sequence1_aligned.sam
samtools view -bt chr11.fa.fai -o sequence2_aligned.bam sequence2_aligned.sam
samtools sort sequence1_aligned.bam sequence1_aligned_sorted
samtools sort sequence2_aligned.bam sequence2_aligned_sorted
samtools merge merged_sequence.bam sequence1_aligned_sorted.bam sequence2_aligned_sorted.bam
samtools sort merged_sequence.bam merged_sequence_sorted
samtools index merged_sequence_sorted.bam
samtools pileup -f chr11.fa -c NG8_s_merged_sequence_sorted.bam > final_alignment.pileup
A run without using data from both lanes produced a similar unusable output.
Any help would be appreciated.
Thanks for reading.