Illumina Guidelines

Services for the Illumina HiSeq 2500 and 4000

The GHTF offers single read, paired-end, and multiplex sequencing using the Illumina HiSeq 2500 and 4000 systems. GHTF offers 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.

Guidelines for Sample Submission for 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

  • 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 RiboZero depleted libraries we require 1ug of total RNA with a 260/280 ratio of 1.7-2.0. 
  • ChIP DNA: Users must prepare their own immuno-precipitated DNA.  We require at least 10ng 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.
  • 16S Metagenomic Library:  Please provide 5ul of samples at a concentration of 5ng/ul in 96 well plate format.
  • Nextera XT Library: Please provide 3ul of samples in a concentration range of 0.1-0.3ng/ul in 96 well plate format.

Guidelines for Illumina Sequencing Sample Submission

Please provide us with a minimum of 10 uL of 10 nM sample in Qiagen buffer EB (Tris-Cl 10mM, pH 8.5.  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.

  • Agilent Bioanalyzer - Illumina recommends using the Bioanalyzer to estimate final product size of the library.  Optimal fragment size range for single end runs is 150-300bp and for paired end runs it is 250-500bp.  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.
  • 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.
  • 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.
Assay Read Length  
Mammalian Genomic DNA 2 x 100 bp  


1 x 100 bp or 2 x 100 bp  
Small RNA 1 x 50 bp  
ChIP-Seq (assuming 1% human genome capture) 1 x 50 bp  
Sequence-Capture (Whole Exome) 2 x 100 bp  

Strategies to mitigate Illumina HiSeq 4000 Index mis-assignment

The integrity of multiplexed samples on Illumina HiSeq 4000 instruments and those with similar chemistry and flow cells as related to index mis-assignment can vary significantly and has been observed to be impacted by the amount of free adapter/primer present in the library, the storage conditions of the library and the library preparation workflow.  Samples most affected by index mis-assignment are low diversity and/or low input such as single cell, circulating DNA or FFPE. 

Illumina recommendations during library construction:

  • Avoid excess adapter/primer by optimzing the PCR or ligation step and then perform extra clean ups
  • Dilute PCR primers to adjust the insert to adapter/primer ratio
  • Perform extra clean ups of each individual library (PAGE, purification colums, Ampure beads)
  • Store individual libraries at -20oC and pool prior to sequencing

Illumina recommendations for pooling of libraries:

  • Use unique dual index pairs
  • Sequence or store individual libraries at -20oC as soon as possible

For applications sensitive to mis-assignment, sequencing on the HiSeq 2500 is available at GHTF.   Libraries constructed/received at GHTF are stored at -20oC and individual libraries are multiplexed just prior to sequencing.