problem to aligning FASTQ through BWA aligner

Question

[modarzi]

Dear All, I need to setup BWA for alignment. I followed below steps:

1-downlod reference genome version through 'wget' and below link:

Wget https://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/chromFa.tar.gz

2-then extract the downloaded file:

tar zvfx chromFa.tar.gz

3- 'cat' .fa file to wg.fa :

cat *.fa > wg.fa

4- remove additional files:

rm chr*.fa

5- Now, I have to generate an index file through the below command by bwa version 0.7.17-r1188:

./bwa index -p hg19bwaidx -a bwtsw wg.fa

After that in the folder I have these 5 files:
hg19bwaidx.amb, hg19bwaidx.ann, hg19bwaidx.bwt, hg19bwaidx.pac,hg19bwaidx.sa

6- Now, I would like to generate a SAM file for a paired-end through mem algorithm based on the below command:

./bwa mem  -T 19  ?????  file_paired_1.fastq  file_paired_2.fastq  > aln.sam

My question is that which one of the five files generated in the indexing process(step 5) should I use instead of ????? at step 6?

I appreciate it if anybody shares his/her comment with me.

Best Regards,

[Pierre Lindenbaum]

3- 'cat' .fa file to wg.fa :

cat *.fa > wg.fa

if you want to use GATK in your downstream analysis you should care about the order of the chromosomes. GATK raises an error if the chromosomes are "chr1" "chr10" "chr11" ... instead of "chr1" "chr2" "chr3" ....

[modarzi]

Thank you for your comment. honestly, I need a BWA-generated SAM file as input for finding CircularRNA in the CircularRNA finder. up to know, I couldn't find any CircularRNA in my samples. could you please guide me on what should I do instead of using the below command:

cat *.fa > wg.fa

Answer 1

[ATpoint]

./bwa mem  -T 19  hg19bwaidx  file_paired_1.fastq  file_paired_2.fastq  > aln.sam

Just the basename of the index of the file.

Usage: bwa mem [options] <idxbase> <in1.fq> [in2.fq]

Pro tip, be safe and do:

find -maxdepth 1 -name "*.fa" | xargs cat > wg.fa

Otherwise it might happen that the shell will interpret the generated output file as an additional input file and will append the output to itself.

[modarzi]

thank you for your comment. Now, which file indicates reference genome? wg.fa or hg19bwaidx?

Answer 2

[andres.firrincieli]

Just use the prefix hg19bwaidx