Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • ZWB
    replied
    I've been doing 16S amplicon sequencing lately and I've found that quantifying by Qubit and pooling works fine, although not quite as good as qPCR. On most runs I see only a slight (+/- 2%) variation in number of reads from any particular barcode indicating that the multiplexing worked well. Given the extra time and cost of running the qPCR, I don't think it's necessary because it only gives slightly better results. For anything other that amplicon sequencing though I would say that quantification by qPCR is an absolute requirement.

    Leave a comment:


  • cmuletz
    replied
    I have been using qPCR to quantify molecules/ul of my amplicons. I have not done a test to see how close Qubit readings are to that of qPCR. I was told by lab members that qPCR is much more sensitive. I typically run ~30 samples on one 454 junior plate and have to do at least 2 qPCR runs to quantify all the samples. I do 3 dilutions for the first run (1:2000, 1:4000, 1:8000) to figure out what dilution I really quantify my samples. This of course depends on PCR cycles and starting DNA amount, but I usually use 1:4000 and 1:8000 dilutions to quantify. BUT I do not use the dilutions to do the emulsion PCR. Most of the time I pipette around 1 ul from each sample, and do one large dilution of that sample. Sometimes 2 ul into 20 ml! I have read and heard from others that they just use Qubit readings and it works fine.

    Leave a comment:


  • Brunda
    replied
    Hello Carly, how did you resolve your problem? I just stumbled onto thread on http://seqanswers.com/forums/showthread.php?t=14511

    i wonder if it's possible to do the qubit measurement instead of QPCR (to be sure you have enough sample) and then going for the norm kit.

    the advantage of QPCR is that it "counts" only dsDNA fragments with both adaptors ligated, so you can be sure it is specific.

    Leave a comment:


  • cmuletz
    started a topic Qubit versus qPCR

    Qubit versus qPCR

    Hello,

    My question relates to the use of Qubit to qPCR for quantifying your amplicon samples to equimolar ratios -- we have both a Qubit/dsDNA HS Assay Kit and qPCR/ Kapa kit in our lab to quantify 16s rRNA amplicon samples with fusion primers. I will pool ~30 samples together. From looking at the previous threads people recommend qPCR and that is the same with the folks in my lab over Qubit and Qubit over nanodrop. However, most microbiome papers I have found people are using Qubit to quantify before pooling. If most people are doing this then...?

    I'm asking for accuracy and time efficiency.

    1) How do you feel about Qubit versus qPCR?

    2) For qPCR are most people doing absolute qPCR over relative? -- I'm not sure if you need the Kapa Kit to do relative qPCR (you would still need to quantify some samples at some point though).

    --relative seems faster with the accuracy -- you have the qPCR results but you don't need to go through the dilutions and replicates to do the absolute qPCR. For absolute qPCR I've been told that you need to do a few dilutions and at least 2 replicates and you need the standards so you can really only quantify 10 samples at a time. I realize with relative you would still need the reps.

    --for my 30 samples that is 3 runs on the machine for absolute versus 1 for relative. In the end I will be doing 4 more pools like this.

    Any folks with experience your comments would be greatly appreciated.

    Thanks!

    Carly

Latest Articles

Collapse

  • seqadmin
    Exploring the Dynamics of the Tumor Microenvironment
    by seqadmin




    The complexity of cancer is clearly demonstrated in the diverse ecosystem of the tumor microenvironment (TME). The TME is made up of numerous cell types and its development begins with the changes that happen during oncogenesis. “Genomic mutations, copy number changes, epigenetic alterations, and alternative gene expression occur to varying degrees within the affected tumor cells,” explained Andrea O’Hara, Ph.D., Strategic Technical Specialist at Azenta. “As...
    07-08-2024, 03:19 PM
  • seqadmin
    Exploring Human Diversity Through Large-Scale Omics
    by seqadmin


    In 2003, researchers from the Human Genome Project (HGP) announced the most comprehensive genome to date1. Although the genome wasn’t fully completed until nearly 20 years later2, numerous large-scale projects, such as the International HapMap Project and 1000 Genomes Project, continued the HGP's work, capturing extensive variation and genomic diversity within humans. Recently, newer initiatives have significantly increased in scale and expanded beyond genomics, offering a more detailed...
    06-25-2024, 06:43 AM

ad_right_rmr

Collapse

News

Collapse

Topics Statistics Last Post
Started by seqadmin, 07-10-2024, 07:30 AM
0 responses
25 views
0 likes
Last Post seqadmin  
Started by seqadmin, 07-03-2024, 09:45 AM
0 responses
201 views
0 likes
Last Post seqadmin  
Started by seqadmin, 07-03-2024, 08:54 AM
0 responses
211 views
0 likes
Last Post seqadmin  
Started by seqadmin, 07-02-2024, 03:00 PM
0 responses
193 views
0 likes
Last Post seqadmin  
Working...
X