We are going to do RNA-Sequencing using Illumina HiSeq for 200 samples. Given that the sample size is fixed, and the budget is fixed, the following 3 options were proposed.
- 50bp pair-end reads, sequencing each sample per lane --> we will get ~100 million reads per sample
- 75bp pair-end reads, sequencing two samples per lane --> we will get ~50-60 million reads per sample
- 100bp pair-end reads, sequencing four samples per lane --> we will get ~30-40 million reads per sample
Based on your experience, which option is the best or you have other suggestions? We would like to do different kinds of analysis for these data, i.e.,novel transcripts, lncRNA, and splicing, SNP, etc. You name it. If we have to sort them by priority (from high to low), I would like to say " novel transcripts, long-noncoding RNAs splicing and differential expression".
Currently, the majority of labs sequence 100bp pair-end, right? But I was told that even you sequence 100bp long, after 75bp, the sequencing quality is very bad due to the issue of sequencer itself, that is, it has nothing with the RNA quality of samples. If this is true, why is 100bp read length becoming more popular now?
Many thanks, Shirley