BioMicroCenter:DNA LIB

From OpenWetWare
Jump to navigationJump to search



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
NEB Ultra II LM-PCR 100pg - 100ng 100nt-500nt
NexteraXT - NexteraFlex Fragmentation with Tn5 transposasE 50ng / 1ng adjustable minimum by SPRI cleanup

NEB Ultra II

Service Standard Illumina Library Prep
INPUT Clean fragmented DNA
INCLUDED Initial QC by Fragment Analyzer
Library preparation
Illumina QC
External - form
UNIT Per sample

The BioMicro Center utilizes the NEB UltraII kit for standard library preparation. This kit is extremely robust and can make libraries from <1ng of input material. It is also amenable to miniaturization and is available as a high-throughput prep as well. NEB Ultra II is based on standard LM-PCR based chemistry. DNA fragments are repaired, A-tailed and ligated to adapters. The ligated products are then amplified by PCR to add indexes and the i5 and i7 anchors. Completed libraries can be size selected using SPRI beads or BluePippin.

NEBNext Ultra II produces the highest rates of conversion to adaptor-ligated molecules from a broad range of input amounts.


Service Nextera Library Prep
INPUT Clean DNA >500bp
OPTIONS Full Nextera (>50ng) - for highest complexity libraries

Nextera XT (>5ng) - for standard libraries
Nextera Flex (>50ng) - for longer inserts

INCLUDED Library preparation
Illumina QC
External - form
UNIT Per sample

Nextera DNA sample preparation, from 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 ATAC-Seq.

The BioMicro Center offers three flavors of Nextera preps, depending on input and throughput requirements. Original Nextera is the most expensive, requiring 50 ng 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 (1 ng) and, thus, less enzyme making it less expensive, but only crude size fractionation using single-pass SPRI selection can be done. Finally, Illumina recently introduced Nextera FLEX, which attaches the Tn5 transposase to a bead. This provides steric inhibition which increases the size of the inserts in the library. Automated versions of the Nextera XT and Flex are available. For more information about high-throughput Nextera, please look at the High-Throuhgput DNA preparation page. The BMC does not offer ATAC-Seq 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.

Standard Nextera workflow.

Example Data

Nextera 2.png

1. Right: 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 Caulobacter 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.

Nextera 3.png

Lower Right: (From the same experiment) 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.

2. Below: An example of a CNV study prepared with Nextera DNA preparation:

CNV data example.png