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A first look at Illumina’s new NextSeq 500

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  • #46
    I think I see the problem - you're using the wrong file name for the reads:

    bbduk.sh in=interleaved.gz out=trimmed.fq.gz ktrim=r k=23 hdist=1 mink=11 tpe tbo minlen=90 ref=truseq.fa.gz,nextera.fa.gz

    bbmap.sh maxindex=200 in=trimmed.fq.fq mhist=mhist.txt bhist=bhist.txt qhist=qhist.txt qahist=qahist.txt

    ...

    Reads Used: 0 (0 bases)

    Normally, BBMap should throw an exception saying it can't find the input file if it does not exist, so I assume there is an empty file named "trimmed.fq.fq".

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    • #47
      Hi Brian, hi Genomax,

      thanks for the replies.
      Brian - that was just a typo by me, it was the correct file.
      Genomax - I am after stats.
      I'm just on my way into work, 6.30am here, I'll log on again there and have another look at my commands.
      thanks.

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      • #48
        @Elsie: This may sound like a dumb question but have you made sure that your interleaved.fq.gz and trimmed.fq.gz files have contents (i.e. they are non-zero bytes in size).

        Post 3-4 sequences from your interleaved and trimmed.fq.gz files (use zmore or zless).

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        • #49
          Hi GenoMax,

          Your dumb question was, unfortunately, spot on - interleaved is fine but trimmed is tiny - hard to map when there is nothing to map! I'll double check this, thank you.

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          • #50
            I'd be interested in seeing the stderr log of BBDuk... it's implausible that ALL of your reads are 100% adapter.

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            • #51
              Thanks Brian. BBmap is currently running, I'll post the results. By the way, I always want to type bbduck...
              thanks.

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              • #52
                Hi Brian,
                My plots look similar, slightly better than yours, but I have decided to postpone posting them as we are moving to V2 chemistry in a few weeks. I'll redo the plots then and post them. Thanks again for all your help, it is greatly appreciated.

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                • #53
                  Hi guys,

                  Just to take note that the v2 chemistry requires NCS 1.4

                  NCS 1.4 is NOT BACKWARD COMPATIBLE for v1 kits. Better to clear up your V1 kits before proceeding with NCS 1.4 upgrades. Illumina is making our life here difficult since the machine is a shared asset in our institution, hence we have to wait for the entire institute to clear up their v1 kits before we can proceed with the upgrade.

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                  • #54
                    You could technically dual boot to have both NCS versions

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                    • #55
                      Hi all,

                      I have the comparative results now for the same library using NexSeq V1 and V2 chemistry, 2x150bp from a bacteria. V2 looks far better.

                      V1:




                      V2:




                      For V2 chemistry, the measured quality is much higher (particularly for read 1), the quality scores are more accurate, and the base frequencies don't diverge as much. Also, 79% of the reads mapped error-free, up from 50% for V1.

                      I also looked at HiSeq2500 data for the same library and it is similar in quality to the NextSeq V2.
                      Attached Files

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                      • #56
                        Originally posted by Brian Bushnell View Post
                        Hi all,

                        I have the comparative results now for the same library using NexSeq V1 and V2 chemistry, 2x150bp from a bacteria. V2 looks far better.

                        V1:


                        V2:


                        For V2 chemistry, the measured quality is much higher (particularly for read 1), the quality scores are more accurate, and the base frequencies don't diverge as much. Also, 79% of the reads mapped error-free, up from 50% for V1.

                        I also looked at HiSeq2500 data for the same library and it is similar in quality to the NextSeq V2.
                        That's great news! Illumina should thank you for your report.

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                        • #57
                          I finished a quality recalibration tool. It seems to work well on NextSeq data with binned quality scores.



                          That shows two different recalibration methods. Even though method 2 looks more accurate, 1 has a larger range and seems to give better results.
                          Attached Files

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                          • #58
                            Hello,

                            we have one big project (RNA and exome seq) midway, for which we have about half of the necessary NextSeq kits (v1). What do you think, can we switch to v2 in the middle of the project, after the v1 kits have been used up? Do you see a problem in the data analysis part, so the data is not actually comparable as the v2 quality is higher?

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                            • #59
                              I asked the same question to Illumina Tech Support last week. Here's their response.

                              "Besides the changes in the software, there was a significant change in the v2 reagents. We changed the dyes of each base to improve the base call and overall sequencing data. This means that we have changed Q30 tables and other features in the software. Overall we do not recommend to compare data obtained between different versions of software.

                              Depending on which application, experimental design and data analysis workflow you may not have significatnt differences. It is your choice to set up the appropriate controls and technical/biological replicates.

                              The best test is to re-sequence a sample with v2 reagents that you have sequenced before with v1 reagents and compare directly the results. "

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                              • #60
                                @Zwerver: This is going to be a tough decision.

                                You could buy a bunch of V1 reagents but V1/V2 require different versions of NCS so until you go through V1 reagents no one else will be able to use V2 chemistry on that machine.

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