Analysis of factors influencing transcript quantification from RNA-Seq paired ended experiments
Factors that may influence transcript quantification
multiple splice forms
polymorphisms
intron signal (intronic seq)
sequencing errors
alignment errors
annotation errors
Differential GC content across exons
Random exon priming
Positional bias (degradation 5->3')
Fragment length?
read length
multiple splice forms
A gene with 1 isoform is trivial
Concurrent expression of multiple isoforms obviously increases the complexity and uncertainty of read assignment. Particularly when combined with multiple levels of transcript expression
False negatives may occur for splice-regulatory variants as the error is proportional on the number and uniformity of expression levels of isoforms.
Read length
Increasing read length decreases numbers of ambiguously mapped reads. This increases the accuracy of quantification.
Transcript identification accuracy is co-dependent on coverage and read length. For coverage > 10million, read length becomes less important as there are plenty of reads to unambiguously identify transcripts.
Transcript quantification accuracy may increase with read length due to increased numbers of correctly detected splice junctions. For read lengths > 50bp identification and quantification accuracy of junctions does not significantly improve
