Hi guys,
I am not new to NGS/RNA-Seq, but I am new to miRNA sequencing... I want to map human miRNA reads (HiSeq 2500, single end, 50bp) to the miRNA databases (mature and hairpin, I took both from here: http://www.mirbase.org/ftp.shtml; I also checked that the headers of the sequences contain "human", so I don't map against the wrong database). This is the procedure I follow:
(1) using cutadapt, remove Illumina adapters, discard reads that are too short (<17bp) or too long (>35bp) after removing adapters -- works fine, no errors/warnings, removes 5-10% of reads from the initial fastq files.
(2) index the miRNA databases.
(3) map trimmed reads (1) to the indexed databases (2)
However, when I do it using stampy, bwa, bowtie or bowtie2, I get less than 0.5% of reads mapped... I believe I'm doing something wrong at the indexing step or am missing something at the alignment step (duh...). Does anyone have an idea of what I could be doing wrong?
You could find all my commands here:
And I also copy them here:
**Stampy**
> stampy.py -g human_mature_mirna -H human_mature_mirna
> stampy.py -g human_mature_mirna -h human_mature_mirna -M reads.fastq -o alignment.stampy.sam
**bowtie**
> bowtie-build mature_dna_human.fa mature_mirna
> bowtie -l 8 mature_mirna reads.fastq > alignment.bowtie.sam
**bowtie2**
> bowtie2-build mature_dna_human.fa mature_mirna.bowtie2
> bowtie2 -L 8 -x mature_mirna.bowtie2 reads.fastq > alignment.bowtie1.sam
**BWA**
> bwa index -a is mature_dna_human.fa
> samtools faidx mature_dna_human.fa
> java -jar CreateSequenceDictionary.jar REFERENCE=mature_dna_human.fa OUTPUT=mature_dna_human.dict
> bwa aln -l 8 Database_for_mirna/mature_dna_human.fa reads.fasrq > alignment.bwa.sai
I hope someone could help!
Cheers,
Irina
I am not new to NGS/RNA-Seq, but I am new to miRNA sequencing... I want to map human miRNA reads (HiSeq 2500, single end, 50bp) to the miRNA databases (mature and hairpin, I took both from here: http://www.mirbase.org/ftp.shtml; I also checked that the headers of the sequences contain "human", so I don't map against the wrong database). This is the procedure I follow:
(1) using cutadapt, remove Illumina adapters, discard reads that are too short (<17bp) or too long (>35bp) after removing adapters -- works fine, no errors/warnings, removes 5-10% of reads from the initial fastq files.
(2) index the miRNA databases.
(3) map trimmed reads (1) to the indexed databases (2)
However, when I do it using stampy, bwa, bowtie or bowtie2, I get less than 0.5% of reads mapped... I believe I'm doing something wrong at the indexing step or am missing something at the alignment step (duh...). Does anyone have an idea of what I could be doing wrong?
You could find all my commands here:
And I also copy them here:
**Stampy**
> stampy.py -g human_mature_mirna -H human_mature_mirna
> stampy.py -g human_mature_mirna -h human_mature_mirna -M reads.fastq -o alignment.stampy.sam
**bowtie**
> bowtie-build mature_dna_human.fa mature_mirna
> bowtie -l 8 mature_mirna reads.fastq > alignment.bowtie.sam
**bowtie2**
> bowtie2-build mature_dna_human.fa mature_mirna.bowtie2
> bowtie2 -L 8 -x mature_mirna.bowtie2 reads.fastq > alignment.bowtie1.sam
**BWA**
> bwa index -a is mature_dna_human.fa
> samtools faidx mature_dna_human.fa
> java -jar CreateSequenceDictionary.jar REFERENCE=mature_dna_human.fa OUTPUT=mature_dna_human.dict
> bwa aln -l 8 Database_for_mirna/mature_dna_human.fa reads.fasrq > alignment.bwa.sai
I hope someone could help!
Cheers,
Irina
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