I haven't worked with miRNA mapping..( the specie is mouse) It is the first time to do it.
I have read a lot of posting and web-site to map my microRNA data.
Here is what I follow.
1) trim the adapter using cutadapt
When I looked at my fastq file (single-end), read length was 36 bp.. by inspecting the eye-ball test, I could not find my common sequences. (So, I am not sure my fastq file has been already trimed or not . So I kept both versions... )This is my command... (comes from http://www.ark-genomics.org/events-online-training-eu-training-course/adapter-and-quality-trimming-illumina-data)
cutadapt -a CGACAGGTTCAGAGTTCTACAGTCCGACGATC \ -a TACAGTCCGACGATC \ -a ATCTCGTATGCCGTCTTCTGCTTG \ -e 0.1 -O 5 -m 15 \ -o xxx_microRNA_adaprm.fastq xxx_microRNA.fastq
So, I have 2 version fastq files. (xxx_microRNA.fastq, xxx_microRNA_adaprm.fastq)
2) Local bowtie2 alignment of miRNA data
(I generated the reference with bowtie-build directly from the hairpin FASTA file downloaded from miRBase.)
> Bowtie2-build hairpin_mms.fa hairpin_mms.fa
> Bowtie2-build mature_mms.fa mature_mms.fa
> Bowtie2 –local –N 1 –L 16 –x hairpin_mms.fa –U fastq/xxx_microRNA(_adaprm).fastq -S xxxx.sam
The bowtie2 mapping returns the following result.. (for both versions)
I shold have done something wrong...
2849144 reads; of these:
2849144 (100.00%) were unpaired; of these:
2847350 (99.94%) aligned 0 times
933 (0.03%) aligned exactly 1 time
861 (0.03%) aligned >1 times
0.06% overall alignment rate
Could you please help me with this?
Oops, I forgot to convert U to T in hairpin.fa file.. (so stupid about it..) Once I run it, I will confirm the result again..