Paired data in RNA-Seq
The CLC Genomics Workbench supports the use of paired data for RNA-Seq. A combination of single reads and paired reads can also be used. There are three major advantages of using paired data:- Since the mapped reads span a larger portion of the reference, there will be less non-specifically mapped reads. This means that there is in general a greater accuracy in the expression values.
- This in turn means that there is a greater chance of accurately measuring the expression of transcript splice variants. Since single reads (especially from the short reads platforms) will usually only span one or two exons, there are many cases where the expression splice variants sharing the same exons cannot be determined accurately. With paired reads, more combinations of exons will be identified as unique for a particular splice variant.27.1
- It is possible to detect Gene fusions where one read in a pair maps in one gene and the other part maps in another gene. If several reads exhibit the same pattern, there is evidence of a fusion gene.
When counting the mapped reads to generate expression values, the CLC Genomics Workbench needs to decide how to handle paired reads. The standard behavior is this: if two reads map as a pair, the pair is counted as one. If the pair is broken, none of the reads are counted. The reasoning is that something is not right in this case, it could be that the transcripts are not represented correctly on the reference, or there are errors in the data. In general, more confidence is placed with an intact pair. If a combination of paired and single reads are used, "true" single reads will also count as one (the single reads that come from broken pairs will not count).
In some situations it may be too strict to disregard broken pairs. This could be in cases where there is a high degree of variation compared to the reference or where the reference lacks comprehensive transcript annotations. By checking the Use 'include broken pairs' counting scheme, both intact and broken pairs are now counted as two. For the broken pairs, this means that each read is counted as one. Reads that are single reads as input are still counted as one.
When looking at the mappings, reads from broken pairs have a darker color than reads that are intact pairs or originally single reads.
Footnotes
- ... variant.27.1
- Note that the CLC Genomics Workbench only calculates the expression of the transcripts already annotated on the reference.