gDNA and RNAseq Illumina Library Preparation
Library Preparation done by FGL: The FGL generates Illumina-compatible libraries from dsDNA or from RNA (total/mRNA/cDNA). We use the Covaris and Bioruptor Pico for shearing and have the majority of the tedious library prep procedures automated on the IntegenX Apollo 324 robot. We also have high-throughput sample rate types for dsDNA submissions (24+ samples) automated on the BioMek4000 utilizing Kapa Biosystems enzymatic shearing preparatory kits. The HT-prep is offered at ~40% reduced rates to the standard gDNA library prep rate.
New Library Preparation Applications: (contact firstname.lastname@example.org for more information)
Whole Genome MethylSeq (from BS conversion → Library Prep)
Nimblegen Custom Capture + Illumina Library Preparation
Human Leukocyte Antigen (HLA) Genotyping (Analysis included)
Small RNA Library Preparations
Dropseq (coming soon)
User Generated Library Preparation: If you are interested in purchasing your own kits for library preparation, the most commonly used kits are from Illumina (Truseq), NEB, Nugen, Bioo Scientific, and Kapa Biosystems. Please keep in mind that library prep kits do not include the Illumina-compatible adapters (which are sometimes sold separately, or can be synthesized). Universal adaptors with PCR amplified indices, and PCR-free adaptors are available for purchase through the GSL, please inquire with email@example.com.
Do-it-yourself instrumentation is also available with the Illumina Neoprep Automated Library Prep instrument. The FGL also houses equipment available for use by users to aid in sample and library prep: Covaris, Bioruptor Pico, Megaruptor, Qubit, Bioanalyzer, Roche LightCycler 96, Blue Pippin, and the Sage-ELF.
Sequencing on the HiSeq4000
Illumina’s sequencing by synthesis (SBS) technology on the HiSeq4000 and 2500 platforms allows massively parallel sequencing using a reversible terminator-based method that enables detection of single bases as they are incorporated into each cluster. A cluster is a clonally amplified fragment of target DNA that is bound to the Illumina flow cell. A fluorescently-labeled terminator is imaged as each dNTP is added and then cleaved to allow incorporation of the next base. Each incorporation step is a cycle and Illumina runs can be done either as Single-end Reads (SR) or Paired-end Reads (PE), which read one end of the fragment or from both ends, respectively.
The older HiSeq2000 platforms were replaced by the HiSeq4000 due to its optimized clustering chemistry and the physical differences between flow cell types that enable 350M -415M reads per lane in a fraction of the time.
All libraries can be run as either SR or PE of varying lengths, but the researcher’s application will determine the run type required. Some of the most common applications include: de novo sequencing, whole-genome and candidate region resequencing, transcriptome analysis (mRNA-Seq), exome sequencing, small RNA discovery, methylation profiling, and genome-wide protein-nucleic acid interaction analysis (ChIP), and various CRISPR applications. Runs can take anywhere from 24 hours for 50SR to 4 days for 150PE. More information regarding the Illumina technology can be found on their website.
Please visit our submissions page for information regarding sample submission and contact us with any questions. We look forward to working with you.
Sequencing on the HiSeq2500: Rapid Run Mode Only
The HiSeq2500 Rapid Run mode utilizes an optimized chemistry set based off the standard v3 SBS chemistry principles but runs only two lanes per flow cell. Even a 150PE will finish in three days time as compared to 17 days on the HiSeq2500 platform’s High Output Mode. The HiSeq2500 platform’s rapid run mode produces approximately 120M-140M reads per lane and is less expensive per lane than the HiSeq4000 but is drastically more per base. The HiSeq2500 is perfect for libraries that do not meet the concentration requirements of the HiSeq4000 or need to maintain continuity with previous data sets generated on the HiSeq2000/2500 platforms.
Additionally, the HiSeq2500 can process 250PE read lengths. Additional information regarding the HiSeq2500 platform’s rapid run mode can be found on Illumina’s website.
Sequencing on the MiSeq
The GSL houses 2 Illumina MiSeq systems, which offer longer read lengths and reduced throughput compared to the HiSeq platforms. The MiSeq can be run in SR or PE mode with customizable read lengths ranging from 50bp to 300bp per direction depending upon the chemistry version. Version 2 chemistry (up to 250PE) on average yields between 12-15M reads while version 3 chemistry (up to 300PE) yields ~25M reads. The MiSeq is perfect for pilot experiment and projects covering smaller target regions or amplicons. Please contact the Director for guidance with amplicon experiment design BEFORE generating amplicons or if custom sequencing primers are required.
Shana McDevitt (Facility Director)
B206 Stanley Hall
Berkeley, CA 94720-3220
(510) 666-3372 (Office and Lab)