aligning miRNA-seq reads to other short sequence data

[impeachme2]

What is an appropriate program for aligning miRNA-seq reads to a collection (multi-fasta of hundreds) of sequences of varying size (ranging from 20 to 200)?

I tried blastn but it didn't seem to like using short sequences as a reference (no hits).

Thanks,

Dan

[aggp11]

You could try bwa's aln-samse/sampe module or the Shrimp2 aligner.

[yangjianhunt]

Hi, Dan,

I recently was doing a similar project to align sRNA deepseq reads to miRNAs, using blastn. The alignment worked really well. But I was aligning short reads to only miRNAs.
My code for blastn is:
blastn -query input.fa -task 'blastn-short' -db 'plant-mirna29-db.db' -word_size '18' -out 29miRblast-$faln -outfmt '6'

Note: you need to specify " -task 'blastn-short'
Also you may want to look at the --help page for all the parameters etc.

I can share my code as following:
# I am processing multiple miR-seq files using the shell script
# I am a beginner so the code below is perhaps not really elegant
code .sh file as follows:

#!/bin/bash


#-----cutadaptors--------

clear
echo "cutting adaptors"
FILESadapt=`ls -1 *.fastq` #note: make sure there is no space in file names! also make sure to convert suffix from fq to fastq.
for fadapt in $FILESadapt
do
echo "cutting adaptors for $fadapt
starting at time: `date +%H%M%S`:"
cutadapt -a TCGTATGCCG --match-read-wildcards -m 9 -o trimmed-$fadapt $fadapt > summary-$fadapt # note: make sure adaptor seq is correct!
echo "adaptors cut for $fadapt at time: `date +%H%M%S`"
done

# ------converting to fasta; collapsing identical reads-----------------
echo "Hi, $USER ! I will use FASTX_tolls to convert fastq files to fasta files and then collapse identical reads now for all trimmed fastq files in the folder"

PATH=$PATH:/home/jiany/Software/NGS-Tools/bin/ # set up the path for fastx_tool
echo "$PATH is set"

FILES=`ls -1 trim*.fastq`
for f in $FILES
do
echo "converting fastq to fasta file for: $f"
echo "start at: `date +%H%M%S`"
fastq_to_fasta -r -n -v -i $f -o $f.fasta
echo "fasta file created for $f.fasta"
echo "finished at: `date +%H%M%S`"
done

#------------------------------

FASTA=`ls -1 trim*.fasta`
for fa in $FASTA
do
echo "collapsing identical reads for: $fa"
echo "start at: `date +%H%M%S`"
fastx_collapser -v -i $fa -o collapsed-$fa.fasta
echo "reads collapsed for collapsed-$fa.fasta"
echo "finished at: `date +%H%M%S`"
done

echo "all completed!!"
echo "All fastx_tools processed at: `date +%H%M%S`"


#----------------blastn to align collapsed reads to 29 plant miRs------------------------

echo "Hi, $USER ! Now I will use blastn to align all collapsed reads to 29 plant miRNAs in the folder"

PATH=$PATH:/home/jiany/blastn-project/ncbi-blast-2.2.26+/bin/
echo "$PATH is set"

makeblastdb -dbtype 'nucl' -in 'mir-q29.fa' -input_type 'fasta' -title 'plant-mirna29-db' -out 'plant-mirna29-db.db'
echo "blastdb for 29 plant miRs created!"

FILESaln=`ls -1 collapsed*.fasta`
for faln in $FILESaln
do
echo "blasting collapsed reads in fasta file for: $faln"
echo "start at: `date +%H%M%S`"
blastn -query $faln -task 'blastn-short' -db 'plant-mirna29-db.db' -word_size '18' -out 29miRblast-$faln -outfmt '6'
echo "Reads aligned to 29 plant miRNAs for $faln"
echo "finished at: `date +%H%M%S`"
done
echo "blastn alignment for all files are done!!"


#----------------formatting result: parsing read counts; sorting on align length and plant miR; add header -------
echo "formatting the blastn table result: parsing read counts, sorting based on plantmiR and aligned length"

FILESfmt=`ls -1 29miR*`
for ffmt in $FILESfmt
do
tr '-' '\t' < $ffmt > table-$ffmt
sort -t $'\t' +3 -4 +5nr -6 table-$ffmt > sorted-table-$ffmt
echo -e "seqID\tseqCount\tSpe\tmiR\tPerAln\tLenAln\tMisMtch\tNumGap\tQstart\tQend\tSstart\tSend\tEvalue\tEvalue2\tBitscore" >> Header-sorted-table-$ffmt
cat sorted-table-$ffmt >> Header-sorted-table-$ffmt
mv Header-sorted-table-$ffmt Header-sorted-table-$ffmt.xls

done
echo "All tasks completed! creating tar ball of result: `date +%y%m%d%H%M%S`-result.tar"
tar -cf `date +%y%m%d%H%M%S`-result.tar *.xls