BioMicroCenter:DNA LIB

From OpenWetWare

Jump to: navigation, search

Image:BioMicroCenter-header6.jpg

The BioMicro Center offers a broad variety of methodologies for preparing DNA libraries for sequencing. As will all of our standard prep methods, pricing includes quality control prior and following prep, along with at least one re-prep for samples that fail. For large batches of samples or 16S/amplicon, please visit our High-Throughput DNA library preparation page.

Method Summary Input Insert Range
SPRIworks LM-PCR 100pg - 100ng 150-350, 250-550 or 100+
Neoprep LM-PCR 1ng - 100ng 250+ only
Nextera / NexteraXT Fragmentation with Tn5 transposase 50ng / 1ng adjustable minimum by SPRI cleanup



Contents

SPRI WORK

Automation by the SPRIworks
Automation by the SPRIworks

Fragmented samples submitted to the BioMicro Center are processed using the Beckman Coulter SPRIworks. This system preforms the sample cleanup, a overhang, ligation and preliminary size selection needed for creating Illumina libraries. The system accepts all amounts of insert and has been extensively tested.

The SPRI-te Nucleic Acid Extractor is a fully-automated library preparation system for Illumina sequencing and supports all Illumina applications except for smallRNA-Seq. It performs all of the traditional Illumina protocol steps listed on the Illumina Library Preparation page with the exception of PCR enrichment. A total of 10 libraries may be prepared simultaneously in 5 hours.

BASICS

The SPRI-Works system consists of three main components: the SPRI-TE Nucleic Acid Extractor, a method card that contains the liquid handling program, and a cartridge containing all of the reagents required to prepare a single library.

The platform utilizes AMPureXP DNA-binding magnetic beads to purify fragmented DNA and perform size selection, eliminating the need for column purification and gel-based size selection. After the adapter ligation and size selection steps are complete, samples are ready for PCR enrichment and clean-up to complete the library prep process. For more information on the SPRI-Works system please visit www.spriworks.com.

SPRIworks
SPRIworks

Size Selection

One of the more remarkable aspect of the SPRIbeads is their ability to provide size selection. SPRI beads are supplied in very high salt/PEG buffer that is needed for their binding. At low amounts of salt and PEG only large molecules are bound to the beads. As more salt is added, smaller fragmetns become bound to the DNA. This allows the "Double SPRI" size selection. A low amount of salt+beads is added to the mix and the high molecular weight material is discarded. Then additional beads (and salt) are added and a smaller size cut is taken with the beads while the low molecular weight material is discarded. On the SPRIworks, there are three size range options available, 200-400bp, 300-600bp, or no size selection. (These ranges apply to the inserts together with the partial adapters.)

Library Complexity

In order to test the performance of the SPRI-te, the same ChIP-seq sample underwent preparation for Illumina sequencing by both an experienced technician and on the SPRI-te. The numbers of unique sequencing reads and significant peaks were doubled in the SPRI-te samples when compared to the manual preparations.

Effect of Concentration

SPRIworks prepared samples have produced high-quality data even when sample input was below the manufacturer's recommendations. To test the dynamic range, we loaded varying quantities (200ng, 50, 12.5, 3.2, 0.8 and 0.2) of 50bp ladder from New England Biolabs on the SPRIworks to produce libraries. We then measured yield using qPCR and the Agilent BioAnalyzer. Results showed a consistent yield between 10% and 30% for the 200bp insert band consistent with the SPRI beads not being limiting.
Constructing Illumina libraries from 50bp ladder
Constructing Illumina libraries from 50bp ladder
Yield of library constructions as measured for the 200nt band
Yield of library constructions as measured for the 200nt band
Relationship between input material for SPRI and library complexity
Relationship between input material for SPRI and library complexity
Experience with the SPRIworks has enabled us to build up an understanding of the relationship between material submitted for the library and the complexity of the library. To the left is data from RNAseq libraries prepared for Sidi Chen of the Sharp lab. The linear correlation between number of molecules sequenced and the input is clearly visible. High complexity libraries required 1ng of input.

Sample Submission

We suggest that you submit as much volume as possible since the first step of the protocol brings all sample volumes up to 100uL in water. For optimal yield, libraries should be fragmented so that the average fragment size is approximately 60nt shorter than the chosen size range to allow for the addition of partial adapters.

All samples submitted for BMC Fragmented DNA sample prep (SPRI-only) must be submitted with user supplied adapter mix. Adapter mixes should be pre-diluted and should have enough volume for 10uL of adapter per sample. For recommended dilutions for various sample concentrations, please see the chart below:

Please note that SPRI-only samples will need to be re-submitted separately if you wish to sequence with us later on.


NEOPREP DNA

The Illumina NeoPrep is the newest library preparation instrument in the core. While the protocols on this instrument allow little room for deviation, the hands off automation should allow for more reproducible library construction.

The Illumina NeoPrep is suitable for large insert fragments for DNA library construction using standard LM-PCR based methods. Testing in the BioMicro Center finds a minimum input of 1ng with all fragments larger than 300bp being incorporated into libraries. Attempts to circumnavigate the size to allow smaller inserts more suitable for ChIP have not been successful. Using Truseq adapters limits input concentrations to 1ng with robust libraries generated with 10ng or more of insert based on testing with a 100bp DNA ladder (NEB).

NEXTERA / NEXTERAXT

Nextera DNA sample preparation, from Epicentre technologies (now a subsidiary of Illumina), is our preferred method for preparing Illumina libraries from intact DNA. Nextera uses a modified TN5 transposase to simultaneously fragment intact genomic DNA and tag it with Illumina adapters. A limited number of PCR steps are required to generate complete Illumina libraries. This very simple method makes this preparation very popular for automation and in vivo methods such as ATACseq.

The BioMicro Center offers three flavors of Nextera preps, depending on input and throughput requirements. Original Nextera is the most expensive, requiring 50ng of input material but produces the most complex libraries. Products can be sized by pippin and are suitable for all applications. NexteraXT uses less input material (1ng) and, thus, less enzyme making it less expensive, but only crude size fractionation using single-pass SPRI selection can be done. Finally, in November 2014, we introduced an automated version of the Nextera XT kit that is done on our Tecan EVOs. For more information about high-throuhgput Nextera, please look at the High-Throuhgput DNA preparation page. The BMC does not offer ATACseq as that works directly with the intact cells.

Nextera works well with amplicons as well as gDNA. However, because two hits are required per molecule to create productive libraries, the ratio of reagent to fragment must be altered significantly to produce good libraries and large inserts may not be possible.

Nextera NexteraXT
Input 50ng 1ng
Sizing Pippin or SPRI SPRI

Example Data

Nextera-prepared samples provided a similar quality of sequencing data compared with samples prepped in parallel on the SPRI-Works system. DNA from the same Colobacter sample was either sonicated for SPRI-prep or provided as intact DNA for Nextera prep. The samples were multiplexed and sequenced together on an Illumina GAII 40bp single-end lane. The total genomic coverage for both Nextera and SPRI-te samples was exactly the same at 97.8% coverage of Colobacter’s GC-rich genome, although complexity was greater in the sonicated samples.


The Nextera transposase does exhibit a mild GC-insertion bias, shown by the increase in percent of the first few bases. Generally, this has a minimal impact on sequencing coverage, though some variation will occur. Below is an example of a CNV study prepared with Nextera DNA preparation:

Personal tools