I have a reads which are quite short (~22nt), and I want to find which mapped to intron-exon boundaries.
I have run tophat with segment=10 but did not find any spliced reads - probably impossible, so something must be wrong with the options I'm using?
Also tried bowtie2 --local, the option I do not see any clipped aligned reads (at least I cannot see in the IGV, is there a way to count the number of trimmed reads that are aligned in --local mode if any?)
•
written
by
manekineko
1 answer
Hi manekineko, I've done simulations with 21 nt hairpin derived reads for rice and human microRNAs and found that even with 1bp indel, the reads are either unmapped or a large proportion of spurious mappings with 16 different mappers. Because performance was somewhat better for the smaller genome, you could get better results by mapping directly to a library of exon junctions.
In addition to Brian's suggestion of BBmap, I would recommend trying
- Bowtie2 with
-very-sensitive-localparameter - Bowtie2 with
-very-sensitiveparameter - Bowtie1
--best --strataparameter - SMALT with
k=10 s=1parameters
•
written
by
mark.ziemann
Log in to answer this question.
-
Different version of Tophat gives me different number of mapped reads
written by BioDH •
-
error while running tophat ReadStream::getRead() called with out-of-order id#!
written by Kritika
-
RNA seq NGS data analysis
written by poonam.bi01 •
-
Counting reads per codon
written by fire_water
-
Tophat does not map reads in some locations on reference genome but bowtie does
written by heshamgibriel •
-
Visualization Split Reads mapped with Segemehl
written by daron.6 •
-
Problems with STAR mapping output
written by manekineko
-
Way to check if there is local trimmed alihned reads BOWTIE2
written by manekineko
-
tophat reporting output problem
written by pavanraaz •
-
Error RNA-seq data, TOPHAT
written by David_emir
Try reducing the seed length, the read has to be longer than the seed length for the alignment to work. Default seed lengths are around 20.
you have no option for seed in tophat or am I wrong? this option is the segment one and I already decrease it to 10
bowtie is the aligner that users the seed lengths - it takes seed length via -L if you use it via tophat the option is called --b2-L I think.
20bp reads are too short for useful spliced alignments unless the introns are extremely short, on the order of a few dozen bp. That said, BBMap can map such reads spliced, with a command like this:
k=10and slow are optional but recommended for mapping such short reads spliced. You can also add the "local" flag but that may reduce the number of reads spanning an intron.