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  • Aligning Illumina paired-end data with read length 250 bp

    Hi All,

    I am quite new in this forum and newbie bioinformatician. I have a very basic question to ask. I have illumina paired-end genomic data with read length 250 bp. I tried some alignment tools to align and map the reads with the reference genome.

    At first I used Novoalign. But I found out that it supports maximum read length 150 bp. Though I posted this problem to Novoalign's forum and they advised me that -n 250 would work. It didn't work actually.

    Then I was exploring BWA. However, BWA only supports maximum read length 200 bp. On the other hand BWA-SW supports more read length, which I think maximum 1000 kbp or something. But BWA-SW doesn't support pairea-end data.

    Then I tried Stampy and it seems taking ages to align and map the data. It took 30 hours and eventually got killed by the system. I think it would not be a wise idea to use a too that takes more than 30 hours to align and map.

    I am now at my wits end. Can anyone please tell me, which aligner I can use to align and map paired-end data with read length 250 bp?

    Many thanks in advance.

    Opulcy

  • #2
    BWA-SW supports paired-end reads. The online page needs to be updated. I would say a better choice is BWA-MEM available from the bwa gitbub, which will be release in the next couple of days (or even today if I can finish the final testing). The released bwa-0.7.0 has a bug that may cause segmentation fault in rare cases.

    Bowtie2 is also a good choice, though as the developer (so could be biased), I think bwa-mem is more accurate. Here is the ROC curve (PDF) for those who are familiar with the metric.

    EDIT: bwa-0.7.1 has been released. It fixes a few bugs in 0.7.0.
    Last edited by lh3; 03-08-2013, 04:17 PM.

    Comment


    • #3
      Aligning Illumina paired-end data with read length 250

      Have you tried Bowtie2?

      Comment


      • #4
        Thanks guys. Your help is much appreciated. I will try BWA-MEM and let you know if anything comes up.

        Regards,
        Opulcy

        Comment


        • #5
          @Ih3

          I ran BWA-MEM and it worked wonderfully as expected. Thanks a lot for your help.

          Regards,
          Opulcy

          Comment


          • #6
            Originally posted by lh3 View Post
            BWA-SW supports paired-end reads. The online page needs to be updated. I would say a better choice is BWA-MEM available from the bwa gitbub, which will be release in the next couple of days (or even today if I can finish the final testing). The released bwa-0.7.0 has a bug that may cause segmentation fault in rare cases.

            Bowtie2 is also a good choice, though as the developer (so could be biased), I think bwa-mem is more accurate. Here is the ROC curve (PDF) for those who are familiar with the metric.

            EDIT: bwa-0.7.1 has been released. It fixes a few bugs in 0.7.0.
            Not being aware of this, um.. I aligned 250bp reads with BWA 0.6.2 and I did not spot any errors or anything out of the usual when aligning 100bp reads. Are my alignments then valid at all? I got an insert size which corresponds to the library prep and all...

            :/

            Comment


            • #7
              Originally posted by lorendarith View Post
              Not being aware of this, um.. I aligned 250bp reads with BWA 0.6.2 and I did not spot any errors or anything out of the usual when aligning 100bp reads. Are my alignments then valid at all? I got an insert size which corresponds to the library prep and all...

              :/
              It is not about being valid or not. You can use bwa-backtrack, the first algorithm, for 250bp reads, but bwa-mem will be better.

              Comment


              • #8
                Originally posted by opulcy97 View Post
                Hi All,

                I am quite new in this forum and newbie bioinformatician. I have a very basic question to ask. I have illumina paired-end genomic data with read length 250 bp. I tried some alignment tools to align and map the reads with the reference genome.

                At first I used Novoalign. But I found out that it supports maximum read length 150 bp. Though I posted this problem to Novoalign's forum and they advised me that -n 250 would work. It didn't work actually.

                Then I was exploring BWA. However, BWA only supports maximum read length 200 bp. On the other hand BWA-SW supports more read length, which I think maximum 1000 kbp or something. But BWA-SW doesn't support pairea-end data.

                Then I tried Stampy and it seems taking ages to align and map the data. It took 30 hours and eventually got killed by the system. I think it would not be a wise idea to use a too that takes more than 30 hours to align and map.

                I am now at my wits end. Can anyone please tell me, which aligner I can use to align and map paired-end data with read length 250 bp?

                Many thanks in advance.

                Opulcy


                Dear Opulcy,

                The Subread aligner can align reads of up to 1200bp long, and it can map paired-end reads. It's performance in mapping 200bp reads has been demonstrated in its paper : http://nar.oxfordjournals.org/conten...kt214.abstract

                Subread is extremely fast. It only takes about 40 minutes to map 10 million pairs of 250bp reads using one thread. Subread can be downloaded from: http://subread.sourceforge.net

                Cheers,
                Wei

                Comment


                • #9
                  Hi Shi,

                  Thanks for such a good information. I will have a look at it.

                  Opulcy

                  Comment


                  • #10
                    Hi,

                    Novoalign Version 3 supports reads up to 1000bp

                    Colin

                    Comment


                    • #11
                      Hi,

                      Also, if you use Tophat2 which includes Bowtie you can use the parameters for whatever read length you may be interested in.

                      Comment


                      • #12
                        BWA mem

                        Hi everybody,
                        I got this error from my first try using bwa mem. Is it a problem of memory?

                        [M::main_mem] read 82646 sequences (10000166 bp)...
                        Segmentation fault (core dumped)


                        Thank you very much for your time

                        Comment


                        • #13
                          Segmentation faults are often a memory problem, although the number of sequences quoted in the error message doesn't seem like a lot.

                          How much memory does the genome index take up?

                          Do you have a small test data set that you can run, or try running the first 1000 reads from your sample to see if that works.

                          Comment


                          • #14
                            This normally means that the program is trying to access memory that isn't allocated/isn't allowed. This may be a deeper problem than just memory usage. Could have to do with accessing files/memory.

                            Are you using the latest version?

                            Comment


                            • #15
                              I'm using the 0.7.5a version and I want to map on the human genome g1k_V37. The index generated a couple of files and the biggest is around 3 gb.

                              Comment

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