QB3 Berkeley / Facilities / Core Research Facilities / QB3 Genomics / Submissions / Sequencing of Illumina Libraries

Sequencing of Illumina Libraries

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All sequencing libraries must be submitted with accurate sizing and quantification. Additionally, qPCR quantification must be ordered for each tube containing Illumina libraries sent to us (except some submissions with >60 samples as detailed below).

Please send 12ul at 10nM or greater in 1.5ml low-binding tubes. Normalization is not required.

Library Sizing

Bioanalyzer, Tape Station, Fragment Analyzer, or Femto Pulse results must be uploaded with your submission, or this service must be ordered per sample. Gel images are not accepted for sequencing libraries. Please submit traces with the x-axis in bp NOT seconds. If possible, please also place a smear range around your library and generate a molarity calculation with the analysis software. Label your samples clearly and with the same ids on the traces and submitted tubes (see recommendations below).

Library Quantification

Sample concentration should be measured with Qubit, Bioanalyzer, or Tape Station, or this service must be ordered per sample. Please provide your concentration in nM if sizing is known.

Extra QC fees will be incurred for libraries greater than 100nM unless the concentration is noted or quantification services are requested.

Buffers

Submit samples in water (or Tris-HCl) where possible, especially if submitting low concentration samples that may need to be concentrated before sequencing.

Labels

Label tubes on the TOP and sides of the tubes.

We strongly recommend the following naming scheme:

  • Name the pool of all libraries being multiplexed together with PI initials, your initials, and a unique numbe (e.g. DRMC001)
  • Name each library within a pool by adding a letter the pool name (e.g. DRMC001A, DRMC001B, etc).

    • User-Pooled Library Submissions

    Please provide sample names and barcodes for all libraries within your pool/s on the sample details sheet when you create your submission. Provide the concentration and sizing of the pool for all sample rows in the sheet (copy & paste across all rows), and likewise use the same “pool name” for all samples that are multiplexed into the same tube.

    GSL Pooled Library Submissions

    The guidelines above apply to each tube containing a library sent to us.

    Indicate which libraries aught to be pooled together by giving them the same “pool name” in the sample details sheet when you create your submission. If pooling equimolar, please indicate a pooling ratio of 1 for all samples. Otherwise, indicate what ratio each library should be within the final pool. Very low concentration libraries can be “spiked” in at a predetermined volume upon request.

    Normalization is usually done by qPCR quantification, so expect a qPCR fee for each tube containing a library sent to us. However, large submissions can be normalized by a MiSeq Nano test run. This method incurs a flat fee for the MiSeq Nano lane. Lastly, we can pool based on user provided concentrations, but will charge a small labor fee per sample and will not repeat uneven lanes.

    Custom Primers

    The GSL is NEVER responsible for failures related to custom sequencing or indexing primers. The user is responsible for primer design. It is very obvious when a run fails due to an unannealed custom primer. Custom primers should be provided to the facility at 100um in 10-20ul aliquots. HiSeq4000 can only accommodate R1 custom primers.

    Sample delivery & drop off

    During COVID restrictions:
    please contact the VC GSL (gsl-fgl@berkeley.edu)

    B206 Stanley Hall (Basement Level 2)
    M-F 10am-5pm

    Shipping Samples to GSL (Sequencing Only, Library Prep goes to FGL)

    Please ship your samples on dry ice overnight to the following address:

    UCB-QB3-GSL
    B206 Stanley Hall
    Berkeley, CA 94720-3220

    If you have any questions regarding sample submission, please contact Christopher Hann-Soden or the lab directly.