Submissions

Submitting RNA

Sample Requirements

General Requirements:

  • Include a DNase treatment with the RNA isolation method - this ensures sample purity and accurate quantitation
  • Samples must be stored in nuclease-free ultrapure water or low TE buffer (10 mM Tris-HCl, pH 7.0-8.0, 0.1 mM EDTA) at -80°C
  • RNA samples must not contain > 1 mM EDTA and must be free of salts and organic contaminants, such as phenol and ethanol since these substances can interfere with library preparation reactions
  • Provide spectrophotometric absorbances from a NanoDrop or similar. A260/A280 and A260/A230 absorbance ratios indicate RNA purity. Values outside these ranges indicate the presence of contaminants
    • A260/A280 ratio of >2.0
    • A260/A230 ratio of 2.0-2.2+

See Evaluating Nucleic Acid Quality for NGS for more information

Illumina Stranded Total RNA Prep with Ribo-Zero Plus, Ligation:

  • 1 - 1000 ng high-quality purified total RNA (RIN ≥ 9) is required in a final volume ≤ 10 μL
    • 10 - 100 ng low-quality RNA (RIN ≥ 2) or FFPE (DV200 > 55%) samples
    • 10 ng minimum for optimal quality & FFPE samples
  • Failure to treat with DNase or inefficient DNase treatment can result in a significant fraction of intergenic reads in the sequence data

Illumina Stranded mRNA Prep, Ligation:

  • 25 - 1000 ng high-quality purified total RNA (RIN ≥ 9) is required in a final volume ≤ 25 μL

Quantification

  • Proper quality control and quantitation requires at least 4 μL of each sample in addition to that required for library preparation
  • Each RNA sample is first evaluated on the Agilent TapeStation 4150 with either RNA or HSRNA ScreenTapes
  • If the samples are of good quality and quantity, the samples will be quantified by Qubit and diluted appropriately for library preparation
  • If the samples are of poor quality or quantity, the customer will be notified with recommendations before moving forward with library preparation

Submitting DNA

Sample Requirements

General Requirements:

  • Samples must be stored in nuclease-free ultrapure water or low TE buffer (10 mM Tris-HCl, pH 8.0-8.5, 0.1 mM EDTA) at RT, +4C, or -20°C
  • DNA samples must not contain > 1 mM EDTA and must be free of salts and organic contaminants, such as phenol and ethanol since these substances can interfere with the tagmentation reaction and result in assay failure
  • Provide spectrophotometric absorbances from a NanoDrop or similar. A260/A280 and A260/A230 absorbance ratios indicate DNA purity. Values outside these ranges indicate the presence of contaminants
    • A260/A280 ratio of 1.8-2.0
    • A260/A230 ratio of 2.0-2.2+

See Evaluating Nucleic Acid Quality for NGS for more information

Illumina DNA Prep, Tagmentation:

  • 1 - 500 ng is required in a final volume ≤ 10 μL
    • 100 - 500 ng minimum for human DNA samples and other large complex genomes
    • 1 ng minimum for small genomes (e.g. microbial)
    • PCR amplicons must be > 150 bp - tagmentation cannot add an adapter directly to the distal end of a fragment; to ensure sufficient coverage of the amplicon target region, design primers to extend beyond the target region by 50 bp per end

Illumina DNA Prep PCR-free, Tagmentation:

  • 25 - 2000 ng high-quality purified DNA is required in a final volume ≤ 25 μL

NEBNext Ultra II FS DNA, with Fragmentation:

  • 0.1 - 500 ng is required in a final volume ≤ 26 μL
  • DNA should be in 1X TE (10 mM Tris pH 8.0, 1 mM EDTA), however, 10 mM Tris pH 7.5–8, low EDTA TE or nuclease-free water are also acceptable

NEBNext Ultra II DNA, without Fragmentation:

  • 0.5 - 1000 ng is required in a final volume ≤ 50 μL
  • DNA should be sheared in 1X TE and diluted with with 10 mM Tris-HCl, pH 8.0, 1X TE, or 0.1X TE

Quantification

  • Quantification by spectrophotometry is only useful as a ballpark estimate for input into a fluorometric assay

Submitting Individual Libraries

The Core is not responsible for failed runs, poor run quality, nor resulting differences in sample representation due to library incompatibility or inaccurate quantitation.

Sample Requirements

  • All libraries to be quantified and sequenced must have Illumina-compatible p5 and p7 sequences and must use the standard NextSeq 1000 read and index primers
  • Evidence the libraries are of suitable size (~250-700 bp average) and molarity (> 4 nM) to be sequenced must be provided
  • The library prep method (including which commercial kit or whether custom primer sequences were used) and indexes of each library must be provided
  • All libraries to be sequenced in a single run can be pooled if they are of the same preparation type and each library sample has unique and compatible indexes

Quantification

  • Each library is first evaluated and quantified on the Agilent TapeStation 4150 with either D1000 or HSD1000 ScreenTapes
  • Each library is quantified by Qubit 1x dsDNA HS assay or by qPCR on the Roche LightCycler 96 using an Illumina-compatible library quantification kit
  • A 2 nM dilution is then made of each library using Illumina-provided resuspension buffer (RSB w/ Tween) and equal volumes are combined into pools for sequencing

Submitting Pools

The Core is not responsible for failed runs, poor run quality, nor resulting differences in sample representation due to library incompatibility or inaccurate quantitation.

Sample Requirements

  • Each library in the pool must satisfy the aforementioned requirements for submitting individual libraries
  • Pools must be at least 2 nM in concentration and a volume greater than 25 μL must be submitted
  • Pools must be prepared with Illumina-provided resuspension buffer (RSB w/ Tween)
  • Each library sample contained in the pool must have unique and Illumina-compatible indexes, preferably unique dual indexes (UDIs)
  • The library prep method and indexes of each library must be provided

Quantification

  • Pools can be quantified by Qubit 1x dsDNA HS assay before loading on the sequencer
  • If the customer declines and provides their own quantification, the Core is not responsible for poor run quality nor resulting differences in sample representation

Submitting Single-Cell/Nucleus Suspensions for 10X

Please see Library Preps - Single Cell for more information about sample preparation and submission requirements.