Look at Different Locus in the Bam File

Question

[santos48]

Hello everyone, I have human RNA-seq data that are reading by MiniON. I have many questions first, when I aligned all FASTQ files with the reference sequence I can not look at the human genome, do I have to align with the hg38 Reference genome for the look at the human chromosome?

If yes why I have a reference file from the reading? and Also let say I aligned with hg38 of my Fastq file, the nanopore sequencing accuracy 20 percent Will this give true results?

Should I align with a specific chr or location in hg38 for correct analysis?

If you have tutorial or article please share with me thank you.

Answer 1

[Devon Ryan]

1. Normally you would align against the most recent version of the human reference genome and tell IGV or IGB or whatever genome viewer you prefer to use that. Then you can visualize the BAM file (assuming you sorted and indexed it).
2. You don't rely on single reads, you rely on the totality of the reads to give you significantly higher accuracy. Use a variant caller for this to make your life easier.
3. Use the entire thing, not just a single chromosome or location. Doing the latter will result in false-positive alignments.

[santos48]

Thank you for answer