Entering edit mode
4.9 years ago
SarahWang
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0
Hello there,
I'm struggling with how to map a ~100 bp FASTQ reads to a 10k shRNA library. I've used bowtie2 --local, seems fruitless. My 10k shRNA library already combined into fa FASTA file, with >shRNAid tag and shRNA sequence. Every time I map, there is 0 alignment. Anyone know how to do that? Really appreciated! Thanks!
What is the length of the shRNA reads? Aren't they shorter than 100bp? Did you try with blast?
SarahWang : Do you have a specific adapter added to these reads that needs to be clipped before you try these alignments? You may also want to try
bowtie v.1if you want to do ungapped alignments.I can trim off the adapters, but not all the fastq reads have the same adapters. And after trimming still not aligned correctly. I'll try bowtie v.1.
I don't know specifically about shRNA but generally in case of small RNA if you don't have the right adapter (which is ligated on 3'-end) then that likely is not a read you are going to be interested in.
Hi h.mon, the shRNA reads are 20bp.
How many sequences in the fastq file? Hundreds, thousands, or millions? It seems you should use the fastq as reference, and map the shRNa to it.
Do you really need to map (that is, you need to know the position), or do you need to filter reads from the fastq that match sequences from the shRNA? If you just need to filter, I suggest bbduk.sh from the BBTools package.
Surely something is not right? Why did you sequence 100 bp if you expect shRNA reads to be 20? Info I looked up actually said that shRNA sequences are 80 bp in length, unless you only captured a part?
The 100bp fastq reads are extracted with target shRNA. I can also use sgRNA, the point is to count number of hits to see which the shRNAs/sgRNAs are related to the phenotype difference.