Information on submitting samples prior to Illumina HiSeq 3000 and MiSeq sequencing

  • Sample drop-off location is in 3012 Agricultural and Life Sciences (ALS) Building, Monday - Friday, 8:00 - 5:00.
  • Optimum library fragment size (insert + adapters) ranges from 150-500bp depending on desired read length and library prep type.  The Agilent Bioanalyzer works well for checking the final library fragment size.
     
  • 260/280 ratio of library should be around 1.8 (NanoDrop).
  • Very important: It is the researcher’s responsibility to accurately quantify and determine the size distribution of the final Illumina prepped sample.  This will allow us to optimize the number of reads and maximize the quality of the data produced.  We recommend using multiple QC techniques.  For quantification, qPCR is the most accurate followed by fluorometric (Qubit) measurement, bioanalyzer, and Nanodrop.
    1. NanoDrop spectrophotometer - Detection limit is 2ng/uL, making it inaccurate for quantifying final diluted product (10nM).  The NanoDrop reading will overestimate the library concentration because it detects all DNA (even w/o adapters), RNA, proteins, free nucleotides, and primers too.  In addition, salts and organic compounds can affect readings.
    1. Agilent 2100 Bioanalyzer – The Agilent Bioanalyzer can be used to estimate final product concentration and to check the size distribution of the library.  The CGRB Core Facilities offers Bioanalyzer service on high sensitivity DNA chips for this purpose.  The detection limit on these chips for DNA sequencing libraries is 100pg/uL.
    2. Fluorometer (eg. Qubit) - This uses fluorescent dyes specific to dsDNA, RNA, ssDNA, or proteins for detection.  More sensitive than NanoDrop so that it can detect concentrations as low as 10pg/uL.
    3. Real-time/quantitative PCR - This protocol uses primers that match the adapters, so that only templates with both adapters will be measured.  The CGRB Core Facilities offers quantification by qPCR using the KAPA Biosystems library quantification kit.  See service fees for costs.  We will need ~12uL of ~10nM library to perform qPCR quantification.  In addition, the CGRB Core Lab houses an Applied Biosystems 7500 Fast Real Time PCR instrument that is available to users after a brief training.
  • Molar concentration of library. The following is a sample calculation:

Library concentration = 40ng/µL
Median size fragment = 170bp
Library molecular weight = 170bp × 650g/mol per bp = 110500g/mol
Molar concentration = 40ng/µL / 110500ng/nmol = 0.000362nmol/µL
= 0.362µM
= 362nM

  • For the concentration to be run on a flow cell it is recommended to run a range to optimize the number of clusters formed. If the DNA concentration is too low then too few clusters are formed and sequencing throughput is low. If the DNA concentration is too high then the density of clusters is too great and complicates data analysis, often filtering many of the reads out.
    For the HiSeq 3000, we typically load 2-3nM with 1% PhiX spike-in for high diversity libraries and at least 10% PhiX spike-in for low diversity libraries.
    The optimum cluster density on the MiSeq for libraries with even base distribution (high diversity) is 1,000-1,200K clusters/mm2 using v.2 kits and 1,200-1,400K clusters/mm2 using v.3 kits. For low diversity libraries we aim for 800 - 1000K clusters/mm2 with 10% PhiX spike-in for both versions of kits.
  • Please provide us with 30uL (if possible) of 10nM sample in Qiagen buffer EB (Tris-Cl 10mM, pH 8.5) supplemented with 0.1% Tween-20 (optional).  We recommend that a final quantification be performed by the Core lab with qPCR.  The sample will then be diluted to the final loading concentration with solutions provided in the Illumina sequencing kits.