What is the minimum read length (Illumina RNASeq data) for alignment using tophat?

More Rakesh Sharma's questions See All

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RNA-seq DEGs downstream analysis and reporting in literature?

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What is the right way to conjugate two acids?

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Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

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Runs of nucleotides in Sanger sequencing?

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Calculating volume of DNA required from DNA concentration?

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Rakesh Sharma

I have reference genome and using it as a guide to do alignment. Now, what is the minimum size of reads, I should consider?

Jon Bråte

I have mapped smallRNAs between 18-24 nts without a problem. But it depends on what you are sequencing and your library size. If you sequenced mRNAs from a library of 300 nt fragments, then short reads (e.g. < 40 nts) could be from degraded mRNAs and not the real genes. So you should set a size cut-off during read trimming.

Fritz J Sedlazeck

You want to have 100bp. Everything below might lead to issues with mappability. Especially if you are having a repeat rich genome or a de novo assembly.

In addition, Tophat might not be the best choice as it is very sensitive to SNPs. Ie. does not map reads in highly heterozygous sites. I think STAR or GSNAP might be a better fit and are faster.