[joseph]

Dear kmcarr,
yes I know that mirbase has the sequences and GFF coordinates but they are multifasta sequences format and not a single sequence file (I am thinking to Mapview that requires a unique fasta sequence...).
I will try to use Bowtie and SAM tool to align and view the sequences and I do not know which format they require.
Do you have an idea?

Thanks and bye for now.
Andrea

Andrea,
have you tried bowtie to map the reads to miRbase as you said? If you did, can you share your findings?

[bioinfosm]

Can people share experience using http://mirexpress.mbc.nctu.edu.tw/usage.php
Seems many of you have your own versions from scripts ..

[kolja]

Hi,

is there a nice tool that allows me to collapse identical reads from Illumina Genome Analyzer or FastA files. I have sequencing data from different small RNAs, with 10-20 million reads. What I want to do is find identical reads and cluster them together as well as count the number of reads in each cluster. Is there a tool that can do that?

[yjhua2110]

several tools for deep sequencing-derived small RNA were provided in deepBase(http://deepbase.sysu.edu.cn/), which were developed to map, store, retrieve, annotate, integrate and visualize deep sequencing-derived small RNAs, and facilitate transcriptomic research and the discovery of novel ncRNAs.

[dukevn]

I use a very similar approach, but I first collapse identical reads before aligning (to avoid aligning the same let-7 and other abundant miRNA reads hundreds of thousands of times. You can then count the number of reads in the original file to generate counts. The only problem with this is that you lose the sequence quality information (if you have a need for that).

Ryan

Hi all,

I have similar question and I found this post from google. Could anybody explain what are identical reads (after trimming I supposed?) and how to collapse them?

Thanks,

D.

[demis001]

Hi all,

I have similar question and I found this post from google. Could anybody explain what are identical reads (after trimming I supposed?) and how to collapse them?

Thanks,

D.

Hi D.

Identical reads are identical sequences. It require simple scripting in order to collapse identical reads; you may write a programme that counts identical reads.

eg. AGCGT
AGCGT
AGCGT
AGAAT
You will write a new file that contains:
AGCGT 3
AGAAT 1

Simple right?

Demis001

[dukevn]

Hi D.

Identical reads are identical sequences. It require simple scripting in order to collapse identical reads; you may write a programme that counts identical reads.

eg. AGCGT
AGCGT
AGCGT
AGAAT
You will write a new file that contains:
AGCGT 3
AGAAT 1

Simple right?

Demis001

Ah, got it now. But I dont get it why we need that counting? Will that do anything with the expression counting after alignment? If they are 3 identical reads, aligning just one is enough, doesnt it?

D.

[demis001]

Ah, got it now. But I dont get it why we need that counting? Will that do anything with the expression counting after alignment? If they are 3 identical reads, aligning just one is enough, doesnt it?

D.

Hi D.,

Yes, aligning one sequence is suffice and saves a lot of time. The expression counting will be done at the end as you said. However, I found that it would be good to store as part of fasta header string in case you might need it down the pipeline. If you are sure you don't need it , don't count at the initial collapse.

eg. >Seq1_$count
TTCCCCGGG

Demis001

[dukevn]

Hi D.,

Yes, aligning one sequence is suffice and saves a lot of time. The expression counting will be done at the end as you said. However, I found that it would be good to store as part of fasta header string in case you might need it down the pipeline. If you are sure you don't need it , don't count at the initial collapse.

eg. >Seq1_$count
TTCCCCGGG

Demis001

I gave it a second thought and even I agree that we can save time by collapsing reads, but we do lose coverage afterward, dont we? Let say in your example, if AGCGT maps uniquely to region R on genome, instead of having 3X coverage (for that read), we get only 1X? But if you count a duplicate number, would it be more difficult to include those numbers after alignment? I think leaving reads as they should be will solve that coverage issue.

[Nomijill]

Really easy to use microRNA tool

Caution: I work for CLC bio

If any of you would like to try a very easy to use tool for microRNA analysis, the 4.0 version of CLC bio's Genomics Workbench includes a start (trimming adapters) to finish (identification of microRNAs in miRBase) analysis pipeline. We have had a tremendously strong response from those who have tried it with both SOLiD data and Illumina data.

If you are interested in giving the MicroRNA tool a whirl, please feel free to download a fully functional trial version. You can also download a tutorial that will walk you through all of the steps. This page has links to both the tutorial and the Genomics Workbench.

http://clcbio.com/index.php?id=1175&cx=007652808978802596904%3Ace7xdfffxvw&cof=FORID%3A11&q=microRNA&siteurl=clcbio.com%2F#165

Whether you like it or not, I am curious to hear what you think.

[olus]

Hi,

is there a nice tool that allows me to collapse identical reads from Illumina Genome Analyzer or FastA files. I have sequencing data from different small RNAs, with 10-20 million reads. What I want to do is find identical reads and cluster them together as well as count the number of reads in each cluster. Is there a tool that can do that?

Hi,
may be FASTX Collapser is what you need

http://hannonlab.cshl.edu/fastx_toolkit/

Cheers
Gabriel

[vkartha]

Collapsing identical reads

I understand how and why the collapsing has to be done, but I'm not sure what to use (even in terms of writing a simple script in python or R) to get the job done, given the files we're talking about are several Gigs in size.

I won't be able to load such a file in R memory for it to work on and collapse based on each read of miRNA 'species' , and obtaining counts for each of them as well.

I thought about using fastx toolkit's collapser program but it wouldn't keep track of the counts which I would need when quantifying mapped miRNAs

Anyone have any ideas? Any help would be greatly appreciated.

[empyrean]

i dont know if you are still looking. but this simple code should do the counts. i got this from this forum only

awk 'NR%4==2' input.fastq|sort |uniq -c|awk '{print $1"\t"$2}'

[mylseq]

Hi all,

I tried to research online but couldn't find solutions related to this. Do you use bowtie to align the reads after it's being collapsed such as in this format?

miRNA-1
AGCGT 3
AGAAT 1

miRNA-101
AGCGTAT 5
AGAATAA 1

It's no longer the conventional fasta format so which option would you recommend using with bowtie? Do I have to make the file to conform to the fasta format prior to using it with bowtie?

Thanks in advance for any help!

M