I am new to Seqanswers (Hi!) but so far have not yet found a helpful thread for my particular problem.
I am trying to do two de novo assemblies of two species of sponges using Trinity. It is an RNA-seq study comparing different tissues and life stages within the respective species, with 2x150 bp reads sequenced to a depth of 124x. There are two whole transcriptomes already published. The GC content of my transcriptome assemblies are ~3-7% lower than the other ones, and GC content falls only after assembly (ie. not after read trimming). Furthermore, my sequences are short (eg. N50 800 vs 1800bp, median length 300 vs 800bp, mean length 600vs 1200bp). The nail on the coffin is that very few reads align as proper pairs (vs individually) - ~50%.
As far as I can tell, there isn't anything about the reads themselves that stand out as red flags, and raw read quality was high.
There are many places where this could have gone wrong, but I would guess it is during assembly. Has anyone received this particular profile before (low GC, many fragments)? If so, what did you do to achieve a better assembly?
Thanks!
I am trying to do two de novo assemblies of two species of sponges using Trinity. It is an RNA-seq study comparing different tissues and life stages within the respective species, with 2x150 bp reads sequenced to a depth of 124x. There are two whole transcriptomes already published. The GC content of my transcriptome assemblies are ~3-7% lower than the other ones, and GC content falls only after assembly (ie. not after read trimming). Furthermore, my sequences are short (eg. N50 800 vs 1800bp, median length 300 vs 800bp, mean length 600vs 1200bp). The nail on the coffin is that very few reads align as proper pairs (vs individually) - ~50%.
As far as I can tell, there isn't anything about the reads themselves that stand out as red flags, and raw read quality was high.
There are many places where this could have gone wrong, but I would guess it is during assembly. Has anyone received this particular profile before (low GC, many fragments)? If so, what did you do to achieve a better assembly?
Thanks!