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  • #16
    Smallest NM value? Sorry - you lost me...

    The idea is to record the hit(s) with the longest identical 5' match(es) to the genome, the theory being that primer artefacts, sequencing errors and RNA edits are all concentrated at the 3' end. We also assume that natural variation is absent for miRNAs. If we get multiple matches with the same score, then all of the matches are recorded.

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    • #17
      Clearly not using NM then!:-) In SAM format, NM = the number of nucleotide differences to the reference sequence. I thought this may be a useful tag for filtering. Or do you just count the length of the match?

      Do you reject sequences longer than 22bp?

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      • #18
        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

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        • #19
          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).

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          • #20
            Originally posted by whsqwghlm View Post
            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.

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            • #21
              Anybody knows how I can trim out the adapter sequence with a certain number of mismatch (1 or 2)?

              Thanks,

              D.

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              • #22
                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

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                • #23
                  Originally posted by didymos View Post
                  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.

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                  • #24
                    fastx_clipper can trim adapter sequence.

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                    • #25
                      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

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                      • #26
                        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!

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                        • #27
                          did you allow mismatches in your alignment ?

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                          • #28
                            No mismatch allowed

                            Originally posted by NicoBxl View Post
                            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?

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                            • #29
                              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 ....

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                              • #30
                                Originally posted by yjhua2110 View Post
                                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?
                                Last edited by JonB; 06-10-2013, 05:26 AM. Reason: lacked quote

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