Very low aligbnment rate for miRNA

Question

[prp491]

I know this kind of question have been asked before but I am not able to solve my problem. I have fastq file containg reads from small RNAseq of non-model plant. I cleaved my adapter (AGATCGGAAGAGCACA) and found peak at 24 nt. I search out the conserved miRNA from miRbase database and predicted the novel miRNA by mirPREFFER. But I got very low percentage (>1% )of alignment when I aligned the my fastq file the predicted miRNAs of my plant. I tried two aligner Bowtie2 and novoalign

I have uploaded the the raw, adapter-trimmed and length sorted fastq files and their respectice fastqc reports HERE

Any help will be much appreciated Thanks

[Rohit]

How long are your sequences? If it is less than 25, it would be the limit for most mappers. Since it is RNAseq could you try segemehl or STAR and see if there are more valid alignments?

[prp491]

I am trying to map 18-24 nt long reads only. Since in literature people have successfully used the Bowtie2 for miRNAseq that's why I used it. I will try to use the START as well as segemehl . Thanks

Answer 1

[harold.smith.tarheel]

Also, check your miRNA reference database to ensure that it contains only a single copy of each unique miRNA sequence. Most miRNAs are encoded by multiple loci and/or are part of highly homologous families, and will be flagged as multi-mappers unless you use the proper reference. You should also specify a shorter seed length for your aligner (per Rohit), and it may also be useful to increase the stringency of alignment (zero or one mismatch).

[prp491]

Here I am confused little bit. Should I used the mature or precursor sequences as reference? My Bowtie2 command looks like this bowtie2 --local -L 15 -N 1

Is it OK? Thanks

[dariober]

+1 For suggesting to tweak the aligner's settings. With bwa mem alignments with score <30 are not returned by default (-T option), this means that even if a match is perfect but too short (say 22bp) you get nothing in output. Again in bwa mem, the minimum seed length is 19 (-k option) which might be too long for miRNA. (I assume bowtie has similar settings to play with).

Answer 2

[igor]

Most RNA-seq tools aren't really designed for very short sequences that you get from small RNA-seq. I would suggest using a miRNA-specific tool, such as:

• miRge - http://atlas.pathology.jhu.edu/baras/miRge.html
• miRDeep2 - https://www.mdc-berlin.de/8551903/en/
• miRExpress - http://mirexpress.mbc.nctu.edu.tw/

[prp491]

Thanks for the advice. miRge is using the Bowtie for alignment that is what I am using for my analysis.

[igor]

Right, but they are also adjusting Bowtie settings.

[prp491]

I+ just tricked the miRge to run with Bowtie2 but I found the same low rate of alignment :(