fastq or bam file for additional alignment

Question

[bioguy24]

Is it better to use a fastq file or a TMAP aligned and unaligned bam and convert those to 2 fastq and combine them? The problem is a fastq is not created by default, but I want to make sure that by converting the aligned then unaligned bam to 2 fastq files then combing them, that will be ok for bowtie2 or bwa-mem or gatk. Thank you?

Answer 1

[Ashutosh Pandey]

Your questions are bit ambiguous. So I am not sure if my answer would be useful. First of all, it seems there is a communication gap between the sequencing core and you. Ion torrent sequencers provide both fastq as well as bam file containing both aligned and unaligned reads unless your sequencing core people have been using some weird default settings. So ask them for the original fastq files. TMAP aligned bam files should work for most of your downstream analyses such as variant calling. You may have to perform some filtering including removing of very short reads aligned by TMAP. It may not necessary though as most of them get lower mapping quality and wont be even considered by variant caller. You may have to modify the bam header in order to make it compatible with GATK. The alternative would be to use TMAP BAM files and use Picard's bam2fastq to create a fastq file. This would give you more control over which aligner you want to use and alignment settings that you want to use.