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  • #1

    single buffer library prep - A-tailing enzyme?

    Hi,
    recently several vendors are offering library prep kits which carry out end-repair and A-tailing without buffer change (simply using a two temperature incubation). The kits do not even require a buffer change/cleanup for the ligation. Please see the links below.

    Would you have any idea which enzymes are used in these kits for A-tailing?

    NEBNext® Ultra™ DNA Library Prep Kit for Illumina® | NEB
    https://www.neb.com/products/e7370-nebnext-ultra-dna-library-prep-kit-for-illumina#tabselect2
    This kit enables DNA Library Prep for Illumina systems, using the original Ultra workflow; Ultra II workflows are now available with improved results.

    http://www.thermoscientificbio.com/ngs-library-preparation-kits/#claseek
    Tags: None
  • #2
    Apparently simply Taq polymerase can be used for A-tailing at very low concentrations:

    Neiman et al 2012:
    Library Preparation and Multiplex Capture for Massive Parallel Sequencing Applications Made Efficient and Easy
    http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0048616
    During the recent years, rapid development of sequencing technologies and a competitive market has enabled researchers to perform massive sequencing projects at a reasonable cost. As the price for the actual sequencing reactions drops, enabling more samples to be sequenced, the relative price for preparing libraries gets larger and the practical laboratory work becomes complex and tedious. We present a cost-effective strategy for simplified library preparation compatible with both whole genome- and targeted sequencing experiments. An optimized enzyme composition and reaction buffer reduces the number of required clean-up steps and allows for usage of bulk enzymes which makes the whole process cheap, efficient and simple. We also present a two-tagging strategy, which allows for multiplex sequencing of targeted regions. To prove our concept, we have prepared libraries for low-pass sequencing from 100 ng DNA, performed 2-, 4- and 8-plex exome capture and a 96-plex capture of a 500 kb region. In all samples we see a high concordance (>99.4%) of SNP calls when comparing to commercially available SNP-chip platforms.


    The published protocol carries out the ligation at 16 degrees overnight, though.

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