DNA Library Preparation Guidelines

The following guidelines apply to those submitting DNA (gDNA, amplified/enriched, plasmid, cDNA) for library preparation. These guidelines are appropriate for libraries being prepared for WGS, metagenomics, ChIP, exome, and various other experiments.

All General Guidelines must be followed in addition to the guidelines specific to Illumina, Oxford Nanopore, or PacBio platforms.

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Sample Delivery Vessel - General Guidelines

  • Place in a 96-well skirted, translucent plate (e.g., Eppendorf twin.tec #00030129300).
    • Sample placement should be going down columns (A1,B1,C1…A2,B2,C2…) in contiguous wells (no skipping wells, rows, or columns unless “Blank” samples).
    • Plate should be sealed tightly with one of the following:
      • If shipping on dry ice, seal wells with tight-fitting 8-cap strips (e.g., Thermo Scientific #AB-0265)
      • If delivering from on campus, firmly seal wells with a clear adhesive seal (e.g., Bio-Rad Microseal ‘B’ Film #MSB1001).
    • Samples that are submitted in other containers will be transferred to a 96 well plate at the submitting lab’s expense. Please inquire for cost of transfer.

Sample Naming Conventions - General Guidelines

  • Each sample must be identified with a simple, unique identifier. If names have been used in previous submissions, don’t repeat in subsequent submissions.
  • The following conventions are strictly enforced:
    • 3-20 characters
    • A-Z, a-z, 0-9, dash (-) are the ONLY acceptable characters
    • Space, underscore, punctuation, and special characters are NOT ALLOWED
    • No replication

Sample Labeling Conventions - General Guidelines

  • All tubes and plates must include the following information.
    • PI name
    • Date
    • Sample name (tubes only)

Sample Quantification - General Guidelines

  • Upon sample receipt, the UWBC will quantify the DNA with a fluorometric assay. QC measures done by the UWBC are considered the gold standard, regardless of the measurements done by the submitting lab. Prior to proceeding with samples that fail to meet these requirements, the lab will be informed.

Illumina Library Preparation - Specific Guidelines

  1. Follow ALL General Guidelines above.
  2. Requires double stranded DNA. Accepted DNA subtypes include gDNA, plasmid, cDNA, amplified DNA, and enriched DNA.
  3. If DNA was quantified using a fluorometric assay (e.g., Qubit), provide 100-500 ng DNA*. Ideal input is 10-100 ng DNA.
  4. If DNA was quantified using spectrophotometer (e.g., NanoDrop), provide 500 ng – 1 µg DNA*. Ideal input is 10-100 ng DNA.
  5. Volume should be 10-50 µl.
  6. DNA should be clean and free of inhibitors. A good indicator of quality DNA is A260/A280 >1.8 and A260/A230 >2.0.

Oxford Nanopore Library Preparation - Specific Guidelines

  1. Follow ALL General Guidelines above.
  2. Requires double stranded DNA (RNase-treatment preferred).
  3. Volume should be 10-100 µl.
  4. Elution buffers TE, Tris HCl, and water are acceptable. EDTA in elution buffers may inhibit enzymes used in library preparation. Provide an aliquot of elution buffer for blank measurement for our NanoDrop measurement.
  5. Verifying purity, size, and mass prior to submission is encouraged but not required. UWBC DNA Sequencing facility performs QC on all input DNA using NanoDrop, Femto Pulse, and Qubit (High Molecular Weight DNA QC) to check for:
    • A260/A280 of ~1.8 and A260/A230 of 2.0-2.2, as measured by spectrophotometer (e.g., NanoDrop).
    • Average fragment size >30 kb, as measured by pulsed-field gel electrophoresis (e.g., Femto Pulse)
    • Input mass of 10 ng to 5 µg (see application specifics below), as measured by fluorometric assay (e.g., Qubit)
  6. Application specific requirements:
    • Whole Genome, Metagenome, Other DNA (BAC/Fosmid*, plasmid*, amplified), Methylation, and Adaptive Sampling require
      • ≥1 µg, ≥25 ng/µl
      • *Circularized constructs must be linearized
    • Cas9 Targeted enrichment requires
      • ≥5 µg, ≥210 ng/µl

PacBio Library Preparation - Specific Guidelines

  1. Follow ALL General Guidelines above.
  2. Requires double stranded DNA (RNase-treatment preferred).
  3. Volume should be 10-100 µl.
  4. Elution buffers TE, Tris HCl, and water are acceptable. EDTA in elution buffers may inhibit enzymes used in library preparation. Provide an aliquot of elution buffer for blank measurement for our NanoDrop measurement.
  5. Verifying purity, size, and mass prior to submission is encouraged but not required. UWBC DNA Sequencing facility performs QC on all input DNA using NanoDrop, Femto Pulse, and Qubit (High Molecular Weight DNA QC) to check for:
    • A260/A280 of ~1.8 and A260/A230 of 2.0-2.2, as measured by spectrophotometer (e.g., NanoDrop)
    • Average fragment size >30 kb, as measured by pulsed-field gel electrophoresis (e.g., Femto Pulse)
    • Input mass of 10 ng to 5 µg (see application specifics below), as measured by fluorometric assay (e.g., Qubit)
  6. Application specific requirements:
    • Whole Genome, Metagenome, Other DNA (e.g., amplified) require
      • ≥5 µg, ≥25 ng/µl
      • Lower inputs may be use, but this may impact optimal loading of SMRT cell