[JonB]

Could you run a program like Cufflinks specifying a mean transcript length of about 18 nts, and then get a gtf file of the different transcripts?

[sBeier]

Usually, with small RNAs being not bigger than 24 nt, you would filter for reads between maybe 15 - 28 nt length. Those are the complete transcripts, so no need for any assembly.

[JonB]

in our deepBase database, we use options: –k 200 –v 0. the Specifying the parameters (–k 200 –v 0) instructs Bowtie to report up to 200 perfect hits for each read.

deepBase is a platform for annotating and discovering small and long ncRNAs from next generation sequencing data. It is available at http://deepbase.sysu.edu.cn

I am mapping reads from a small RNA sequencing, and I am curious how you treat the results after mapping with the -k 200 option? Do you assemble the transcripts? Or do you report all the locations where a read has been mapped?

[unique379]

Dear all,

as above discussions and suggestion, could i use bowtie something like this for miRNA alignment ?? just please make me sure.

bowtie --sam -q -n 0 -l 15 -e 99999 -k 200 -a --chunkmbs 1024 --best ....

[zeam]

No mismatch allowed

did you allow mismatches in your alignment ?

What I want is to dissect the correlation between smRNA and DNA methylation.Previous studies reveals that both of miRNA and siRNA involed in DNA methylation.The problem I met is 1)mapping;2)separating miRNA and siRNA from smRNA-seq data.Can you give me some suggestions if you have some experience on these subjects?

[NicoBxl]

did you allow mismatches in your alignment ?

[zeam]

Hi everyone,
When you were processing smRNA-seq data,which software did you use?I have attempted SOAP2 and bowtie,both of them didn't work well enough,many of the reads could not map to the reference.But when I used blat,it works well except consuming time.Can you guys give me some suggestions?My data is from plants,and I want to sepate miRNA and siRNA from smRNA-seq data.
Thanks!

[jdanderson]

Hello Everyone,

I would like to second mgogol's suggestion. It is part of a larger set of tools called FASTX Toolkit: http://hannonlab.cshl.edu/fastx_toolkit/index.html

I've found the tools to be very useful and user friendly.

Cheers,
Johnathon

[mgogol]

fastx_clipper can trim adapter sequence.

[dukevn]

Hi,

In Bowtie you can trim n nucleotides from 3' or 5' end with -3 n or -5 n option - so you can play with this and -v 2 for two mm. However for trimming I am using blast with smaller word size -W option, and then choosing only those mappings which are on the 3' end.

tomek

Thanks, but I want to trim the adapter sequence, not n nucleotides.

[didymos]

Hi,

In Bowtie you can trim n nucleotides from 3' or 5' end with -3 n or -5 n option - so you can play with this and -v 2 for two mm. However for trimming I am using blast with smaller word size -W option, and then choosing only those mappings which are on the 3' end.

tomek

[dukevn]

Anybody knows how I can trim out the adapter sequence with a certain number of mismatch (1 or 2)?

Thanks,

D.

[yjhua2110]

Each read may map exactly to many places in the genome. We want to capture all of these locations to a threshold promiscuity, typically 100, over which we discard all of the mappings (i.e. if 101 alignments are returned from the search).

you can use the options: -a -m 100. Specifying -m 100 instructs bowtie to refrain from reporting any alignments for reads having more than 100 reportable alignments.

[whsqwghlm]

Each read may map exactly to many places in the genome. We want to capture all of these locations to a threshold promiscuity, typically 100, over which we discard all of the mappings (i.e. if 101 alignments are returned from the search).

[didymos]

I am new in the miRNA field and I am wondering why you are using -k 200 or 101 option? In other words why you want to have 200 alignment with 0 mismatches, rather than one unique with 0 mm?
Thanks!

tomek