Hello! I am new to RNA seq. I am trying to assemble a transcriptome, de novo. I have 100 bp paired end reads. I am having a hard time finding a good guide to paired end de novo assembly; I have been taking bits and pieces from different guides, however, I am starting to become confused. I would like to explain the steps I have done so far, and any advice on things I have done wrong, or need to do, would be greatly appreciated!
- I used fastx toolkits quality trimmer to trim any reads with a phred score of <30.
- I know I am supposed to clip adapter sequences, however, I do not think I have any on my reads? is this possible? is there a way to check?
General question: I have paired end reads... am I supposed to quality trim them separately? By separately, I mean quality trim the left reads and then quality trim the right? No matter how much reading i do on PE reads, I am still confused on this.
3. I used a left read and its corresponding right read to assemble a transcript using trinity. Does anyone know how to tell if i needed to set a strand specific parameter?... such as RF, FR.... and how I figure this out if need be?
I know this is many MANY questions. Any help would be so so so much appreciated!