Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • three-read barcoding and adapters

    Hi all,

    We are intending to multiplex 10 individuals on a single Illumina lane for PE sequencing, and we have access to Multiplexing Primers, Adapters and Primer Indices previously ordered from Invitrogen (not Illumina) for another project. I have gone through the sequences, comparing them to what's been posted on SEQanswers, but there is only a partial match.

    Our Multiplexing Adapters (MA1 and MA2) are:
    MA1: 5' P-GATCGGAAGAGCACACGTCT
    MA2: 5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT

    ...but there is partial disagreement (in the latter half of the sequence) between MA1 and the adapter posted on some threads (but not others), such as the adapter sequence provided by ECO in a post from 7 April 2008:
    5' P-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG

    The same goes for our PCR primer 2
    (5' GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT), for which ECO provides a completely different sequence
    (5' CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT)

    Adapter 1, PCR Primer 1 and the Indices are identical, though, between ECO and us.

    I guess I am only trying to make sure we have the correct adapters, and to understand why there can be different sequences out there for what should essentially be the same adapters/primers. Have there been changes along the way?

    I would also be very thankful if someone could help me out with an additional question:

    For adapter ligation, a mix containing Adapters 1 and 2 needs to be combined with ligase buffer, ligase and the DNA sample. However, what concentrations do the two adaptors need to be in? I wasn't able to find this information anywhere in the Illumina protocol.

    Please excuse the rather lengthy inquiry, and I'd be very thankful for any advice.

    Cheers
    Bjorn

Latest Articles

Collapse

  • seqadmin
    Strategies for Sequencing Challenging Samples
    by seqadmin


    Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
    03-22-2024, 06:39 AM
  • seqadmin
    Techniques and Challenges in Conservation Genomics
    by seqadmin



    The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

    Avian Conservation
    Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
    03-08-2024, 10:41 AM

ad_right_rmr

Collapse

News

Collapse

Topics Statistics Last Post
Started by seqadmin, Yesterday, 06:37 PM
0 responses
8 views
0 likes
Last Post seqadmin  
Started by seqadmin, Yesterday, 06:07 PM
0 responses
8 views
0 likes
Last Post seqadmin  
Started by seqadmin, 03-22-2024, 10:03 AM
0 responses
49 views
0 likes
Last Post seqadmin  
Started by seqadmin, 03-21-2024, 07:32 AM
0 responses
67 views
0 likes
Last Post seqadmin  
Working...
X