I read about the new rlsim package Rlsim, A Package For Simulating Rna-Seq Library Preparation With Parameter Estimation and have some questions regarding efftest where I am mostly interested in getting a number (or some numbers ) for PCR artifact/bias.
I am wondering about the input. The program requires "aligned paired end reads in SAM format" aligned to the transcriptome.
- will it work for single end data?
- what about the non-aligned reads, that could exhibit even more extreme bias. Will they show up in the calculations?
- what about repetitive reads?
1 answer
- The
effesttools works on paired-end reads only as single ended data has not enough information about the sequenced fragments in order to estimate the insert size distribution and GC biases. - No, unaligned reads are not taken into account as we have no information about the transcript of origin.
- I guess by "repetitive reads" you mean multimapping reads. There is no special treatment for multimapping reads, but you can further restrict the set of single isoform transcripts (through the
-iflag) to the set of single isoform transcripts withouth a significant number of multimapping reads.
EDIT: 3'. In the case you mean PCR duplicates by repetitive reads: they are implicitly taken into account during the estimation of PCR biases, as they contribute the increased number of fragments in the GC bins with higher efficiency.
EDIT 2: One more thing regarding multimapping reads: effest filters the input alignments according to the minimum mapping quality specified by the -q flag (10 by default). If your aligner assigns a mapping quality lower than that to multimapping reads, than they are effectively discarded!
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