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  • rudzik79
    replied
    no product

    I tried NebNext and to my surprise I got completely no product while implementing Illumina's protocol (25 cycles in 1st PCR). At first I thought either the mix or my primers were not working. Neither after 1st round, nor after 2nd, index PCR did I get anything.

    I ran it with SYBR Green, and to my surprise really at 25 cycles there was completely nothing. It started being visible at 26-27 cycles and reached plateau at 33-34 cycles. iProof HIFI from BIORAD was exactly the same, for that matter. I did not have enough money to order KAPA, but I tested some samples that were previously successfully amplified on Illumina KAPA protocol.

    So be careful with NEBNext and iProof, you'd better check how many cycles you need first on a qPCR.

    Leave a comment:


  • luc
    replied
    Thanks! We will watch out for potential problems with our Kapa Hifi amplifiations.

    Leave a comment:


  • vandykitty
    replied
    Yes these are poor samples and I have been doing nothing but troubleshooting this project since day 1. After doubling the reaction and cleaning up the samples with Ampure beads, I did achieve a confident Bioanalyzer trace. I sequenced 70 samples using the 'other vendor' reagents for PCR1, Old Kapa for PCR2 (indexing) and achieved 85-99% bacterial composition with 977KK/mm2 so the coverage was more than what was needed. While I do not have a nice summary of all my failed runs, when I used the new KAPA for PCR2 (indexing) I frequently had insufficient input DNA to move forward, so I tired the old KAPA and it worked.

    Leave a comment:


  • luc
    replied
    Hi vandykitty,

    is this not too much background smear, no matter which of the three polymerases?
    Last edited by luc; 05-19-2016, 04:46 PM.

    Leave a comment:


  • vandykitty
    replied
    Reviving this older post possibly. I have been having issues with a new lot of samples. I purchased a new lot of KAPA 2X HiFi MM in March and I was having trouble with both PCR steps. I tested out Bioline HotStart (because it was in the lab) and much less background and stronger products. Following the 16S protocol I was still sticking the with 'New Kapa' kit and the indexing PCR was failing constantly. I finally got out an old batch of KAPA 2X HiFi and it finally worked for my samples!!! I went back to the next batch of samples and clearly show that the New KAPA reagents and they want me to do more troubleshooting, ugh! Really, I have been doing this for over a year and never had a issue before. I will try the Q5/Phusion as suggested above... anyone else have a suggestion?
    Attached Files
    Last edited by vandykitty; 05-19-2016, 05:21 AM. Reason: fiel attachement

    Leave a comment:


  • SSatoshi
    replied
    Originally posted by cheezemeister View Post
    Just an update: We did a run this week where we included replicates of some samples where we replaced the KAPA HiFi with NEBNext Q5. We'll see next week if there are any detectable differences.
    Were there any difference between KAPA HiFi and NEBNext Q5? I'm looking for a good PCR enzyme for my library prep (Tailed-PCR method), and found your thread.

    Leave a comment:


  • cheezemeister
    replied
    Originally posted by jmhopkins View Post
    I'm also unimpressed with the paired 300 kits to begin with but that's another issue.
    If you're talking about the sharp drop in Q scores on the reverse read, we're experiencing that as well. Apparently this is a known issue with the 300 kits, and Illumina has been working on a solution for a while. They suggested to me that I use the 250 kits, but that's really pushing it for getting read merging on the V3/V4 amplicon. The reads would need to be near-perfect full length.

    Leave a comment:


  • jmhopkins
    replied
    It's been awhile since we used the v4 kit. Most of our researchers have turned to v1-3 if they are doing metagenomics. The original v1-3 protocol was pretty dicey and we were seeing lots of failures/strange size artifacts in the second PCR step (indexing). With their updated protocol it was much better but not perfect; however, there were still some unexpected failures. Additionally, sequencing these on our MiSeq took some adjustment as they seem to cluster poorly (likely due to their crazy large size) as well as perfectly good libraries dropping out of sequencing for no real reason. I'm also unimpressed with the paired 300 kits to begin with but that's another issue.

    Leave a comment:


  • cheezemeister
    replied
    Just an update: We did a run this week where we included replicates of some samples where we replaced the KAPA HiFi with NEBNext Q5. We'll see next week if there are any detectable differences.

    Leave a comment:


  • cheezemeister
    replied
    Originally posted by jmhopkins View Post
    Have you tried moving to a completely kitted 16S protocol? We've been using Bioo Sci 16S v1-3 with fairly good success, even with extremely low input samples and highly mixed samples (human/bacteria). They also make a v4 only kit as well. The price is pretty reasonable and you can multiplex up to 384 samples if need be.
    We tried the Bioo v4 kit and weren't happy with it. We couldn't get reproducible amplification; amplicon concentrations were all over the map. And perhaps more important, we couldn't get clean amplicon using their protocol. There were lots of other peaks on the bioanalyzer traces that we couldn't get rid of. We haven't tried the V1-V3 kit.

    That, plus the V3/V4 protocol gives higher resolution OTUs than the V4 alone.

    Leave a comment:


  • jmhopkins
    replied
    Have you tried moving to a completely kitted 16S protocol? We've been using Bioo Sci 16S v1-3 with fairly good success, even with extremely low input samples and highly mixed samples (human/bacteria). They also make a v4 only kit as well. The price is pretty reasonable and you can multiplex up to 384 samples if need be.

    Leave a comment:


  • Chimplint
    replied
    Howdy!

    No RNA-seq experience, but recently started a Genotyping-by-Sequencing (GBS) project. It originally called for Taqman 2x, but I found NEBNext Q5 HF to produce a better sequencing library compared to the Taqman2x and Kapa HiFi.

    Leave a comment:


  • bilyl
    replied
    Originally posted by cheezemeister View Post
    I'm currently doing 16S sequencing using Illumina's protocol which uses KAPA HiFi ReadyMix (KK2602) for amplification. However, the cost of this mastermix is obscene and it makes me sick every time I have to order it. Does anyone know of an alternative mastermix for a bit better price (or a much better price)?

    I see that NEB has dropped their price on NEBNext Master Mix. Has anyone tried this out? What's the performance like, and is it a direct replacement for KAPA?

    Thanks!
    Actually, I'm not sure if anyone has noticed this but I've observed a lot of off-target "hits" when using KAPA HiFi for 16S amplification, especially when there is human host DNA present. Q5/Phusion seems to work quite well.

    Leave a comment:


  • microgirl123
    replied
    I use Phusion HF from NEB, which I think is half the price, for the second round of PCR. I use a different PCR program from the one in the Illumina protocol though and use Nextera (rather than Nextera XT) adapters.

    Leave a comment:


  • cheezemeister
    started a topic Alternative to KAPA HiFi Readymix

    Alternative to KAPA HiFi Readymix

    I'm currently doing 16S sequencing using Illumina's protocol which uses KAPA HiFi ReadyMix (KK2602) for amplification. However, the cost of this mastermix is obscene and it makes me sick every time I have to order it. Does anyone know of an alternative mastermix for a bit better price (or a much better price)?

    I see that NEB has dropped their price on NEBNext Master Mix. Has anyone tried this out? What's the performance like, and is it a direct replacement for KAPA?

    Thanks!
    Last edited by cheezemeister; 07-16-2015, 08:10 PM.

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