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  • Multiplexing RRBS samples

    Dear everyone,

    Im preparing a RRBS library. I want to multiplex 10 samples from 150ng DNA.

    We use ilumnia DNA nano LT adaptors on the fragmented DNA.

    I have been looking online for multiplexing guidelines, but cannot seem to find anything on what is recommended.

    I used these adaptors:
    TRUSEQ_1 ATCACG
    TRUSEQ_2 CGATGT
    TRUSEQ_4 TGACCA
    TRUSEQ_5 ACAGTG
    TRUSEQ_6 GCCAAT
    TRUSEQ_7 CAGATC
    TRUSEQ_8 ACTTGA
    TRUSEQ_10 TAGCTT
    TRUSEQ_11 GGCTAC
    TRUSEQ_12 CTTGTA

    Would this multiplexing with the converted DNA cause too low diversity of clusters or could it be manageable?

    We would spike in with 30% PhiX as allways to reduce cluster diversity.

    We use a protocol similar to this:
    Sequencing-based approaches have led to new insights about DNA methylation. While many different techniques for genome-scale mapping of DNA methylation have been employed, throughput has been a key limitation for most. To further facilitate the mapping of DNA methylation, we describe a protocol for gel-free multiplexed reduced representation bisulfite sequencing (mRRBS) that reduces the workload dramatically and enables processing of 96 or more samples per week. mRRBS achieves similar CpG coverage to the original RRBS protocol, while the higher throughput and lower cost make it better suited for large-scale DNA methylation mapping studies, including cohorts of cancer samples.


    Thank you for your help.

  • #2
    For multiplex sequencing of more than six Nano LT indexes, you do not have to be concerned with index diversity so your ten-plex will be fine. If you want detailed compatibility tables you can find them in TruSeq Nano library prep user guide. Nano adapters have mC so they do not get converted by bisulfite. Obviously, you will need more than one sequencing lane if they are mouse libraries.

    There is a new commercial kit which directly ligates adapters to MspI overhang and includes 0-3 diversity bases. These libraries do not require PhiX spike-in and can be sequenced like standard libraries with 10-20% less cluster.

    Comment


    • #3
      Originally posted by nucacidhunter View Post
      For multiplex sequencing of more than six Nano LT indexes, you do not have to be concerned with index diversity so your ten-plex will be fine. If you want detailed compatibility tables you can find them in TruSeq Nano library prep user guide. Nano adapters have mC so they do not get converted by bisulfite. Obviously, you will need more than one sequencing lane if they are mouse libraries.

      There is a new commercial kit which directly ligates adapters to MspI overhang and includes 0-3 diversity bases. These libraries do not require PhiX spike-in and can be sequenced like standard libraries with 10-20% less cluster.
      Wow, thank you very much for your quick and precise answer.

      Sounds really nice, do you have the name of one of these suppliers?

      Comment


      • #4
        NuGEN Ovation RRBS kit. There is a bit of discussion regarding the kit in this thread: http://seqanswers.com/forums/showthr...rbs#post181746

        Comment

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