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A lot of my reads are not mapped (reads too short). Is there anything I can do to map shorter reads?

The term "too short" in the "% of reads unmapped" category of "UNMAPPED READS" from the "" file refers to the alignment length, not the read length. The average trimmed length before mapping is reported as "average input length". Significant "% of reads unmapped: too short" could either happen for normal-length reads that are not mapping well, or for reads that were too short (over-trimmed) before mapping. Therefore, check the following:

  • Per Sequence GC content: check to see if there are any prominent peaks. If so, this could be an indication of contamination in the library.

  • Overrepresented sequences: Check the origin of the top sequences by copying the sequence and pasting into BLASTn. If the sequences do not align to the species used for library preparation, there may be a contamination issue. A common cell culture contaminant is Mycoplasma.

  • RNA-seq library preparation: ensure appropriate QC is performed on final libraries.

  • Processing data errors: ensure the appropriate species was selected.

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