Illumina Platform Library Preparation and Sequencing

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:

Any amount of additional sequencing can be requested per sample at our Extra Data rate.

Library Preparation Services

All libraries are prepared at the FGL. Library prep related questions should be directed to fgl-qb3@berkeley.edu.

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.

Equipment Sharing

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 the following Illumina sequencers, 2 MiSeqs, and a NextSeq 2000. We also provide additional sequencing options through our various core partnerships.

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.

New users are strongly encouraged to schedule a consultation regarding their project to determine the appropriate sequencing run and parameters.

Sequencing 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

Custom primers are available on our MiSeq and NextSeq 2000 platforms only. Custom primers should be provided to the facility at 100um in 10-20ul aliquots and should be HPLC purified.

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.

Pooling of Sequencing Libraries

We can pool libraries by qPCR quantification or by sequencing on a MiSeq nano kit. Please see our rates sheet for associated costs.

Illumina Library Preps by the FGL

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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 Library Sequencing

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).

Library Volume and Concentration

Please submit 15-20 uL of your libraries at a minimum of 10 nM in 1.5 mL tube in an EDTA free elution buffer or Molecular Biology Grade water (RNase/DNase Free). Please reach out to the lab if you need to submit less than our minimum requirements for advice.

Library Sizing

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.

Library Quantification

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.

Buffers

Submit samples in water (or Tris-HCl) where possible, especially if submitting low concentration samples that may need to be concentrated before sequencing.

Labels

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 number (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

Please provide sample names and barcodes for all libraries on the sample details sheet when you create your submission. Provide the concentration and sizing for each library. Please use the same “pool name” for all samples that are to be multiplexed together. For submissions with a single pool, the pool name will be the same on all rows. If you are sequencing multiple pools in different lanes, please indicate this with multiple pool names.

Custom Primers

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 and should be HPLC purified.