The rates for our Illumina Library Prep and Sequencing Services can be found here.
Full Service Prep & Sequencing By-the-Sample
Our full service pipeline provides the greatest flexibility, cost efficiency, and guarantee of service for most projects. We charge by the sample with no minimum project size, provide our standard library prep, then pool together multiple submissions to sequence on the most economical lanes. Sequencing is 150bp paired-end reads, and any samples that fail to reach the guaranteed target are automatically re-queued. RNAseq projects are re-queued as a single pool to eliminate any batch effects.
The minimum data amounts per sample are:
|RNAseq||25 M reads|
|Human/Mouse/Rat Exome||5 Gbp (100x)|
|Human/Mouse/Rat Genome||12 Gbp (30x)|
Any amount of additional sequencing can be requested per sample at our Extra Data rate.
Library Preparation Services By-the-Sample
All libraries are prepared at the FGL. Library prep related questions should be directed to email@example.com.
DNA and RNA Library Preparation
Most steps of library preparation are automated, so small submissions may face additional wait times while they are collected with other small submissions to be processed in parallel. Low input and PCR-free preps are available upon request with an additional fee or sample requirements, respectively. We can also provide custom preps with advance notice – please schedule a consultation.
DNA samples are sheared using Covaris or Bioruptor Pico ultrasonicators, while RNA samples are heat fragmented. We use KAPA HyperPrep reagents (Roche). For all library preps, we ligate on a stub Y-adapter, which are extended to full length indexed adapters via 1-5 cycles of PCR. All libraries utilize Unique Dual Indexes to eliminate index hopping.
Hybridization Capture, including Whole Exome Library Preparation (Human/Mouse/Rat)
We use the IDT xGen Exome Research Panel for HMR exome capture. Other species or custom target capture experiments are possible, but probes must be submitted along with the samples.
The FGL provides equipment for use by trained UCB researchers to aid in sample and library prep, including: Covaris, Bioruptor Pico, Megaruptor, Qubit, Bioanalyzer.
Illumina Sequencing By-the-Lane
The GSL operates several Illumina sequencers, including 2 MiSeqs, a NextSeq 2000, a HiSeq 4000, and a NovaSeq 6000. We also provide additional sequencing options through our partnerships with UC Davis and UCSF cores.
Sequencing is ordered by-the-lane, but different sequencers and different flowcells have different numbers of lanes, so sequencing formats may have to wait some time unless an entire flowcell is ordered.
|Instrument||Flowcell||Lanes per Flowcell|
|NovaSeq 6000||SP, S1, & S2||2|
New users are strongly encouraged to schedule a consultation regarding their project to determine the appropriate sequencing run and parameters.
Illumina’s sequencing by synthesis (SBS) technology proceeds in cycles, where each cycle corresponds to one base sequenced. Sequencing kits are sold by the number of cycles they support, from “35 cycles” to “600 cycles” in a run. A run is further divided into 2-4 stages, which proceed in the following order: Read 1, Index 1, Index 2 (optional), Read 2 (optional). The nominal number of cycles in a kit only counts the Read 1 + Read 2 cycles, so all kits contain some “extra” reagents for the Index reads.
Sequencing runs with only a Read 1 stage are referred to as Single Read (SR), while runs with Read 1 and Read 2 stages are referred to as Paired End (PE), with Read 2 being on the opposite strand from Read 1 (sequenced in reverse). Index 1 sequences the i7 index and is always sequenced in the same orientation as Read 1, but depending on the platform Index 2, which sequences the i5 index, may be in one orientation or the other.
All properly constructed libraries can be sequenced in either SR or PE formats. We strongly recommend using a Dual Unique Indexing strategy for all libraries, where possible, to eliminate index hopping. When specifying custom run parameters, please provide the number of cycles you require in “R1-I1-I2-R2” notation. For example, a 100bp paired end run with a single 8bp index would be “100-8-0-100”.
Custom Sequencing Primers
Runs with custom primers may or may not be compatible with samples in another lane depending on the primers and on the sequencing platform. Please inquire with us before requesting partial flowcell sequencing using custom primers.
Failed runs due to custom primer design and/or affinity issues are not the responsibility of the facility in ANY situation and users will be charged for failed runs.
Full Service DNAseq and mRNAseq Guidelines (library prep and sequencing)
- RNA-seq – Submit 400ng minimum DNase treated total RNA in 5~50µl ultra-pure water, for mRNA selection (PolyA selection or RiboZero). RNA samples must be accompanied by an Agilent 2100 bioanalyzer profile to determine integrity of the sample (or we can analyze your samples on the Bioanalyzer). Ribodepletion is available on a case-by-case basis.
- DNA-Seq (genomic DNA, cDNA, amplicons, WGBS, Metagenomics, etc)- Submit 200 ng of dsDNA (purified or amprified DNA) in under 50ul of ultra pure water for most amplications, WGBS or PCRfree require 1ug. ChIP samples must be accompanied by QPCR verification. We can make library from any >5ng of dsDNA but extra charges apply for use of extra reagents and higher PCR duplicate rates should be expected. Please contact us first if you have than 1~10ng of DNA.
- Small RNA – Submit 200ng~1ug of total RNA or enriched samples in 10 µl ultrapure water. RNA samples must be accompanied by an Agilent 2100 bioanalyzer profile to determine integrity of the sample. Please note that a Trizol purification method or colums for small RNA preps should be used to retain small RNAs in total RNA.
- Small amount-We are offerring RNA-seq or small RNA-seq libraries from low amount of RNA (1~30ng) as a customized service. We are going to provide this services if there is enough interest. Please contact us(opens in a new tab) if you want to try us out on these.
- Single cell – 10x Genomics Single Cell services are available for local projects. Please contact (opens in a new tab)Justin if you are interested in using this system.
- Please contact us(opens in a new tab) if you do not find your application here. We might be able to help you as a customized collaboration project.
User-Prepped Illumina Sequencing Library Guidelines
All sequencing libraries must be submitted with accurate sizing and quantification. Additionally, qPCR quantification must be ordered for each tube containing Illumina libraries sent to us (except some submissions with >60 samples as detailed below).
|Tube:||1.5ml low-binding tube/s|
Bioanalyzer, Tape Station, Fragment Analyzer, or Femto Pulse results must be uploaded with your submission, or this service must be ordered per sample. Gel images are not accepted for sequencing libraries. Please submit traces with the x-axis in bp NOT seconds. Also, please place a smear range around your library and generate a molarity calculation with the analysis software. Label your samples clearly and with the same ids on the traces and submitted tubes (see recommendations below). For pooled libraries please submit a trace of the final pooled library, not the individual subsamples.
Normalization is not required, but concentration should be measured with Qubit, Bioanalyzer, or Tape Station. Nanodrop quantification is not sufficiently accurate. If you do not have access to an accurate quantification method you can submit your entire prep volume and request us to provide Qubit quantification.
Please provide your concentration in nM if sizing is known. Extra QC fees will be incurred for libraries greater than 100nM unless the concentration is noted or quantification services are requested.
Submit samples in water (or Tris-HCl) where possible, especially if submitting low concentration samples that may need to be concentrated before sequencing.
Label tubes on the TOP and sides of the tubes.
We strongly recommend the following naming scheme:
- Name the pool of all libraries being multiplexed together with PI initials, your initials, and a unique numbe (e.g. DRMC001)
- Name each library within a pool by adding a letter the pool name (e.g. DRMC001A, DRMC001B, etc).
User-Pooled Library Submissions
Please provide sample names and barcodes for all libraries within your pool/s on the sample details sheet when you create your submission. Provide the concentration and sizing of the pool for all sample rows in the sheet (copy & paste across all rows), and likewise use the same “pool name” for all samples that are multiplexed into the same tube.
GSL-Pooled Library Submissions
Normalization is usually done by qPCR quantification, so expect a qPCR fee for each tube containing a library sent to us. For pooling via qPCR please submit samples in individual 1.5 mL tubes at a minimum concentration of 10 nM and minimum volume of 12 uL. Larger submissions (40+ samples) can be normalized by a MiSeq Nano test run. This method incurs a flat fee for the MiSeq Nano lane. Please submit samples in a 96 well plate. Ideally we like a minimum concentration of 1nM, a minimum volume of 40 uL, and for sample concentrations to be normalized to within one order of magnitude. Please contact the lab (firstname.lastname@example.org) prior to submitting projects for nano pooling. Lastly, we can pool based on user provided concentrations, but will charge a small labor fee per sample and will not repeat uneven lanes.
The GSL is NEVER responsible for failures related to custom sequencing or indexing primers. The user is responsible for primer design. It is very obvious when a run fails due to an unannealed custom primer. Custom primers should be provided to the facility at 100um in 10-20ul aliquots. HiSeq4000 can only accommodate R1 custom primers