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A first look at Illumina’s new NextSeq 500

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  • GenoMax
    replied
    @rogerzzw: I assume you have been in touch with Illumina tech support about this? What was their take?

    In general tag read issues with other illumina sequencers (don't have direct NextSeq experience) happen when a sample is (borderline) overloaded. Has that possibility been eliminated?

    Leave a comment:


  • rogerzzw
    replied
    WGBS poor index and read2 on Nextseq

    Hi, Guys

    I am new to NGS. We've been trying to do whole genome bisulfite sequencing with Nugen Kit on Nextseq 500 for a few times with 2 samples.

    We always encountered the same problem. After demultiplexing, there is about 50% of unknown index reads (most of them are "GGGGG" and "NNNNNN") . According to other centers using exact same kit and machine, they do not have any problems with adapters and indexes. And we always use single-use illumina kit and reagent for each run. So we do not know what happens.

    We ve also tried to map read1 and read2 (with unknown barcodes). The alignment of read1 is quite good (90% aligniable), but reads2 is not good (~40%).

    We used custom primer for read1 and illumina primers for read2 and index.

    Do you guys have some ideas on what happened?

    Many thanks in advance!

    Roger

    Leave a comment:


  • SA2002
    replied
    Thanks for the replies. Actually we recently ran kits with v1.3 but noticed that the stickers on the flow cells looked slightly different from previous flow cells and we got low density clusters. We're in the process of trying to figure out whether our reagents were v1 or v2 and was just curious if anyone else ran into the same issue.

    Leave a comment:


  • GenoMax
    replied
    Originally posted by SA2002 View Post
    Hello,

    Has anyone used v2 reagents with NCS v1.3? If so, what did the results look like?
    Quote from here:http://support.illumina.com/sequenci...xtseq-500.html

    NCS v1.4—Required for use with the NextSeq 500 Kit v2. NCS v1.4 is optimized for v2 reagents. Earlier kit versions are not compatible.

    NCS v1.3—Compatible NextSeq v1 reagents.

    Leave a comment:


  • TonyBrooks
    replied
    Originally posted by SA2002 View Post
    Hello,

    Has anyone used v2 reagents with NCS v1.3? If so, what did the results look like?
    I'm pretty sure you can't do this as the software would surely check which kit version you are using and tell you it's incompatible.
    It also looks like they changed the dyes, so the lasers would most likely excite at the wrong wavelength.

    Leave a comment:


  • SA2002
    replied
    Hello,

    Has anyone used v2 reagents with NCS v1.3? If so, what did the results look like?

    Leave a comment:


  • TonyBrooks
    replied
    This upgrade really is a pain in the backside. I have no idea why Illumina couldn't make this back compatible and then use the RFID chips to determine whether to process the run as required.

    I wonder what happens if someone comes wanting to compare to an older data set once we've switched to v2. They'll need to pay to re-run those old libraries on v2.

    Leave a comment:


  • GenoMax
    replied
    @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.

    Leave a comment:


  • TonyBrooks
    replied
    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. "

    Leave a comment:


  • Zwerver
    replied
    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?

    Leave a comment:


  • Brian Bushnell
    replied
    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

    Leave a comment:


  • ymc
    replied
    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.

    Leave a comment:


  • Brian Bushnell
    replied
    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

    Leave a comment:


  • fanli
    replied
    You could technically dual boot to have both NCS versions

    Leave a comment:


  • kentawan
    replied
    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.

    Leave a comment:

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