Further, as the prevalent transcription of RTEs will results in a large population of fragmented transcripts of sizes similar to piRNAs, analysis should be restricted to libraries of RNAs associated with Piwi proteins.
In RNA-Seq analysis of differential gene expression in different conditions, it is very common that de novo assembling of eukaryotic transcriptome often reports fragmented transcripts of one gene or repeated contigs that only differ by a single-nucleotide polymorphism (SNP) or a base insertion or deletion (indel).
The choice of the k-mer size can either lead to fully assembled or heavily fragmented transcripts, changing the quality of an assembly dramatically.
This step was not needed for the Ancistrus library enriched in fast evolving transcripts as the ISSH method results in a PCR product containing fragmented transcripts, generally in the range of 300 to 1000 bp.
Probably, a certain number of transcripts could actually represent alleles, paralogs, fragmented transcripts, spliced forms, immature mRNAs and even a combination of them.
In this procedure, RNA is incubated with calf intestinal phosphatase (CIP), leading to the 5' dephosphorylation of tRNA, rRNA, and fragmented transcripts.
Low-expression genes were assembled more effectively with small k-mer sizes, leading to the assembly of numerous and highly fragmented transcripts, whereas high-expression genes were assembled more effectively with large k-mer sizes, emphasizing contiguity [ 12, 14].
Many of these sequences might represent fragmented transcripts.
Therefore, despite the inevitable presence of broken transcripts, the results of the de novo assembly were extremely satisfying, highlighting that about half of the sequences, contained in the final set of transcripts, was assembled to the full length or very close to it and that just about a quarter of the contigs were resulting from highly fragmented transcripts.
The residual signal observed for each mutant may represent intact or fragmented transcripts, or may have arisen from spurious products formed in the absence of a bone fide target.
Transcripts of less than 1000 nt have a median 1.3 fold higher expression level when comparing unfragmented to fragmented transcripts.
