Illumina Guidelines

Illumina HT DNA Sequencing Guidelines

The GHTF offers single read, paired-end, and multiplex sequencing using the Illumina HiSeq 2000 system. Beginning in September 2010, the GHTF will offer library preparation services for DNA-seq, RNA-seq, and Chip-seq and other standard sequencing libraries. Instrumentation in the GHTF dedicated to DNA sequencing includes a Covaris DNA shearing system, Agilent BioAnalyzer 2100 capable of running the high-sensitivity “DNA Lab-on-a-Chip”, NanoDrop ND-1000 spectrophotometer and QuBit Fluorometer, in addition to standard molecular biological instrumentation. Incoming DNA or RNA samples are checked for quality (Agilent Bioanalyzer 2100) and quantity/concentration levels (NanoDrop ND-1000 spectrophotometer or QuBit Fluorometer) prior to initiation of processing steps. Sequence from the GHTF is piped across the campus 10 GB network to the Institute for Genomics and Bioinformatics (IGB) computer cluster dedicated to sequence processing and data storage. IGB personnel are responsible for converting data to FASTQ files and notifying users when they may download those files via FTP. The IGB also performs basic DNA alignment against reference genomes and RNA-seq analysis as part of the DNA sequencing fee. Additional analysis including DNA assembly is available on a fee-for-service basis.

Library Construction 

We prepare sequencing libraries for paired-end genomic sequencing, RNA-seq, ChIP-seq, barcoded, targeted capture, and whole exome sequencing.   Contact the facility for pricing (949-824-6023 or ucightf@uci.edu).

gDNA: DNA should be provided in TE buffer. We require at least 500 ng with a 260/280 ratio of 1.7-2.0. The 260/230 ratio should be >2.  

RNAseq: Total RNA should be provided in DEPC treated water. For RiboMinus or RiboZero depleted libraries we require 5ug of total RNA with a 260/280 ratio of 1.7-2.0.  For Ambion-NuGen RNA-Seq libraries (human, mouse and rat only) we require 250 ng of total RNA with a 260/280 ratio of 1.7-2.0.  If your RNA is isolated from cultured cells make sure that there is no mycoplasma contamination!  Mycoplasma RNA is not polyadenylated and will show up in your library if you use rRNA depletion and random priming for cDNA synthesis.  

Barcoded gDNA: DNA should be provided in TE buffer. We require at least 500 ng with a 260/280 ratio of 1.7-2.0. The 260/230 ratio should be >2.

ChIP DNA: Users must prepare their own immuno-precipitated DNA.  We require at least 50ng with a 260/280 ratio of 1.7-2.0.  Note the less the starting input of IP-DNA the greater the number of amplification cycles must be done.  Increased cycle numbers leads to an increase in amplification artifacts.

Targeted or Whole Exome Capture: We require 3ug of DNA with a 260/280 ratio of 1.7-2.0. The 260/230 ratio should be >2.  Note there are several different suppliers of whole exome capture reagents.   The GHTF can advise you on the coverage of your gene of interest from different vendors.

Guidelines for Illumina Sequencing Sample Submission

• Optimal fragment size range for single end runs is 150-300bp and for paired end runs it is 250-500bp.
   
• 260/280 ratio of library. Should be around 1.8 to 2.0 (NanoDrop).
• The final Illumina prepped sample MUST be quantified accurately to optimize the number of reads and maximize the quality of the data produced.   The GHTF will run any of the following quantification and QC methods for a modest fee.

We recommend the following quantification and QC methods.

1. NanoDrop Spectrophotometer - Detection limit is 2 ng/uL, making it inaccurate for quantifying final diluted product (10nM).  The NanoDrop reading will overestimate the library concentration because it detects RNA, proteins, and primers too.  In addition, salts and organic compounds can affect readings.
2. Agilent Bioanalyzer - Illumina recommends using the Bioanalyzer to estimate final product size of the library.  The GHTF offers Bioanalyzer service on high sensitivity DNA chips for this purpose.  The detection limit on these chips for DNA sequencing libraries is 100pg/uL.
3. Flourometer (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 10 pg/uL.
4. Realtime Quantitative PCR - This uses primers that match the adapters, so that only templates with both adapters will be measured. This is the most accurate method for determining library quantification.  The GHTF offers Q-PCR quantification as a service.

Molar concentration of library. The following is a sample calculation:

Library concentration =  25 ng/µL
Average fragment size = 300 bp

Library molecular weight = 300 bp × 650 g/mol per bp = 195000 g/mol

Molar concentration =  25ng/µL/195000ng/nmol = 0.000128 nmol/µL = 0.128 µM = 128 nM

• Concentration to be run on flowcell. It is recommended to run a concentration 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 high and the lane could fail.
• Please provide us with a minimum of 30 uL of 10 nM sample in Qiagen buffer EB (Tris-Cl 10mM, pH 8.5). We will dilute the sample to the final concentrations with solutions provided in the CBot kit. 10 nM of a standard library is approximately 1-2 ng/uL, which cannot be accurately measured with the NanoDrop.  We can dilute libraries at a higher concentration.  However, libraries at a lower concentration we will charge to SpeedVac them to a higher concentration.

Recommended # of Lanes Assuming 30x coverage (=99.95% accuracy) for Human Genome