Aligning Reads From Small Rna-Seq Onto Human Genome

Question

[Leszek]

I'm working with human small RNA-Seq (50 bp, single end). Surprisingly, I'm able to align only tiny fraction of reads (<1% for 3 mismatches, <5% for 6 mismatches). I have tested 2 aligners (bowtie and gem-mapper) and got similar results. Do you have any idea why is that?

[Damian Kao]

Have you tried doing a fastqc on the reads? What do the quality look like?

[DG]

Is it possible that you are missing something really simple? What are bowtie's default options for mapping RNA-seq reads on to a reference genome? Are you mapping to a reference genome or exome? You may want to try Tophat which does Bowtie first for short-read alignment but also allows alignment across splice junctions so it can do splice-junction mapping.

[Leszek]

@DK: I trimmed reads at first base having quality <20 and discarded reads shorter than 31 bases @Dan: I'm mapping onto hg18 (bowtie --sam --all -n3 -l21). Optionally hard clipping -5 10 or -3 10

[Jeremy Leipzig]

show us a sequence you think should have aligned

Answer 1

[Nicolas Rosewick]

I'm sure you forgot to trim the 3' adapter ! try trimmomatic, fastx_clipper or cutadapt

[David Langenberger]

When doing small RNA-seq, the main fraction of your reads is around 22-24 nt in length (the miRNA fraction). Without clipping the adapter sequences, there is no way to map most of the reads of your experiment. I would recommend you to clip the used adapter (not only 10 nt) and then take a look at your length distribution. You should see a peak at 22-24nt... if not, there is something wrong with your experiment.

[David Langenberger]

And if you don't know your adapter sequence, hard clip a longer fraction of the 3'end... at least 25nt. Then you should be able to map ~80%, I assume.

[David Langenberger]

By the way: The adaptor for the Illumina HiSeq 2000 miRNA protocol is TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC.

Try this: ./fastx_clipper -i SRR324686.fastq -o SRR324686.clipped.fastq -a TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC -l 15 -M 20 -c

[xiaojuhu13]

I run it as you said , but many miRNA reads trimmed still too long far from 20nt, what should I do next?

[xiaojuhu13]

and the adaptor for the Illumina HiSeq 2000 miRNA protocol is TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC., when I search in miRBase, there appeared some mature miRNA accessions, then how to explain the sequences are adapters or mature miRNA sequences? And why there are different illumina adaptors like TCGTATGCCGTCTTCTGCTTGT(A: Problems with analysis of small RNAseq data - Adapter trimming)?

[Leszek]

I don't thinks so, when I hard clip 10bp from 3' only 11% reads align, clipping 10bp from 5' gives 3% reads aligned.

[David Langenberger]

And if you don't know your adapter sequence, hard clip a longer fraction of the 3'end... at least 25nt. Then you be able to map ~80%, I assume.

Answer 2

[Darked89]

Check the size of the inserts. You may be running into adapter sequences. Mapping with last will give you all unique mappings, assuming you got long enough inserts. Also mapping to mirBASE makes more sense when you have 21bp inserts.

Answer 3

[Vikas Bansal]

If the problem is really because of adapters, then I will recommend MicroRazerS: rapid alignment of small RNA reads.paper. Reads can be of arbitrary length and can contain adapter sequence at the 3' end, you can find rest of the information here.