Full Service Options
DNAseq and mRNAseq (library prep and sequencing)
Full Service Prep NovaSeq submission for available here.
500ng of purified gDNA and/or 500ng of Total RNA are required in ~50ul of EDTA-free buffer with relevant QC for submission (Bioanalyzer/Gel traces, concentration, and nano drop ratios but can provide QC for additional cost).
All samples will be prepared into standard sized libraries using Kapa Biosystems library preparation kits (with covaris/bioruptor shearing for gDNA) and indexes with custom Unique Dual Indexes to eliminate cross sample bleed between resultant Fastq files per sample. Any number of samples is allowable from 1 – 100+ but samples will be pooled together with other users submissions on a 150PE NovaSeq S4 flowcell. Data return is 10Gb for gDNA or 25M read pairs per sample for mRNAseq. Additional data can be requested in increments of 10Gb/~40M read pairs.
Whole Genome and Whole Exome Library Preparation
Human WGS preps are performed with Kapa Hyper Prep reagents and Unique Dual Indexed Y-Adapters with 1 cycle of PCR, the sequenced at 30x coverage on a NovaSeq 6000 S4 150PE lane (~120Gb of data), no minimum sample count requirements. 500ng of gDNA by Qubit or 1000ng of gDNA by Nanodrop must be submitted with agarose gel to show quality of sample (can provide QC for additional cost).
WES preps are performed using the IDT xGen Exome Research Panel at 80-100X with Unique Dual Indexes and minimal PCR cycles, then sequenced in 120 sample batches on a NovaSeq 6000 S4 150PE lane (~5Gb data), no minimum sample count requirements. 200ng of gDNA by Qubit or 500ng of gDNA by Nanodrop must be submitted with agarose gel to show quality of sample (can provide QC for additional cost).
Library Prep Only Services (order sequencing by the lane):
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 an Biomek FXp. We utilize Kapa Biosystems library prep reagents but have Swift and Clontech kist available for low input sample preparations protocols. PCR-free gDNA preps are also available assuming 200ng of gDNA as measured via Qubit. All sample submissions require concentration information and Agarose gels be submitted with the samples to avoid further QC costs. Total RNA must be submitted with a bioanlayzer trace validating RIN score or the facility will process and charge the user. mRNAseq preps require RINs of 8+. Please refer to the FGL website for submission requirements or contact Shana McDevitt with questions. firstname.lastname@example.org
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 always include the Illumina-compatible adapters (which are sometimes sold separately, or can be synthesized). Please not that Unique Dual Indexes should be used for reduced index hopping and barcode bleed between samples. They can be ordered from Illumina and Nugen or are available for free from the GSL for UCB investigators.
The FGL/GSL also houses equipment available for use by trained users to aid in sample and library prep: Covaris, Bioruptor Pico, Megaruptor, Qubit, Bioanalyzer, Roche LightCycler 96, Pippin, Blue Pippin, and the Sage-ELF.
Sequencing Illumina Platforms
Illumina’s sequencing by synthesis (SBS) technology on the HiSeq, MiSeq, NextSeq, and NovaSeq 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 HiSeq2000/2500 platforms were replaced by the HiSeq4000 and NovaSeq due to optimized clustering chemistry and the physical differences between flow cell types that enable far more data per lane or flowcell.
All properly constructed 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 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. The GSL offers v3 kits (15-25M reads) which can be run as 150SR, 75PE, or 300PE. The GSL also offers MiSeq Nano runs (1M reads) for small pilot projects (150PE or 300SR). The MiSeq is perfect for pilot experiments and projects covering smaller target regions or amplicons. Failed runs due to custom primer design and/or custom primer priming issues are not the responsibility of the facility in ANY situation and users will be charged for failed runs.
Shana McDevitt (Managing Director)