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Next Gen Sequencing

Documentation and Support

454 Rapid Library Adapters and Fusion Primers

In an exclusive agreement with 454 Life Sciences, a Roche Company, IDT is able to provide researchers with 454 Fusion Primer design software and custom primers for the Genome Sequencer FLX System.

454 Fusion Primers are required for a number of 454 Sequencing applications. These applications include "ultra-deep", amplicon sequencing for detection of low-frequency mutations, and the comprehensive identification and quantification of common and rare sequences within a population. These studies are easily initiated with 454 Fusion Primer design software to target individual exons or all exons from one or more genes.

Roche recommends IDT’s HPLC-purified 454 Fusion Primers as the best way for a researchers to ensure they are getting the best data from their sequencing runs. As with all IDT oligos, identity is guaranteed with 100% Mass Spec QC.

Roche

GS FLX Titanium Rapid Library Adaptors

Access all available Molecular Identifier (MID) tags available. MIDs will allow identifying tags to be placed on multiple libraries so that they may be multiplexed for sequencing. In an agreement with Roche, IDT provides MIDs 13 - 132.

454 GS FLX Standard and Titanium Fusion Primers

Access all available Molecular Identifier (MID) tags available. MIDs will allow identifying tags to be placed on multiple libraries so that they may be multiplexed for sequencing. In an agreement with Roche, IDT provides MIDs 13 - 132.

GS FLX Titanium Rapid Library MID Adaptor Oligos

Titanium Rapid Library MID Oligos are intended for shotgun sequencing; the MIDs are included in order to multiplex different samples on a single sequencing run.

GS FLX Titanium Rapid Library MID Adaptors Extended List

The GS FLX Titanium Rapid Library MID Adaptors Extended List includes a set of Adaptors that can be substituted for the Adaptors of the GS FLX Titanium Rapid Library Preparation Kit for the preparation of DNA libraries for sequencing on the Genome Sequencer FLX System. Tagging multiple libraries with different MIDs allows them to be amplified and sequenced together in a single region of the PicoTiterPlate device. For customers that need more MIDs than the twelve supplied by 454 in the GS FLX Titanium Rapid Library MID Adaptors Kit, this Extended List provides additional sequences that have been optimized for MID applications.

Each MID sequence contains at least 4 changes (insertion, deletion, or substitution) to make it unique from the other members of the Extended MID Set. This means that, for any of these MIDs, it is possible to either detect up to 2 errors and correct 1 error, or alternatively, detect 3 errors and correct none.

The Extended Set MIDs are sorted according to the number of reagent flows needed to sequence each, with a lower number indicating fewer flows. As a result, the lower numbered entries of the Extended Set MIDs are preferable to the higher numbered Adaptors because they can be sequenced using fewer reagent flows thereby maximizing the number of remaining flows for sequencing the library fragment. 

GS FLX System Workflow

  1. Generate single-stranded template DNA library
    • Short adaptors that are specific to the 3′ and 5′ ends are added to each fragment for the purification, amplification, and sequencing steps. The single-stranded DNA library is then immobilized on DNA Capture Beads and emulsified. The result in one bead per unique fragment.
  2. Amplify the library with emulsion PCR (emPCR)
    • Each unique fragment is amplified individually but simultaneously with the other fragments in the library. The result will be several million copies of each unique fragment.
  3. Sequence to generate data
    • The Genome Sequences FLX Instrument uses a flow of individual nucleotides in a fixed order to sequence the hundreds of thousands of wells that each contain one bead.
  4. Analyze data with bioinformatics tools

For more information on this process, please see the Roche information on How it Works

Overview of Multiplexing

Through the use of unique Multiplex Identifier (MID) Adaptors for each individual library, the libraries can be amplified by emPCR and sequenced together in one multiplex reaction. Once the sequencing reads are complete, the data analysis software can deconvolute the data so that each individual library can be identified by its MID tag.

Application for GS FLX Titanium Rapid Library MID Adaptors

The GS FLX Titanium Rapid Library MID Adaptors can be substituted for the Adaptor of the GS FLX Titanium Rapid Library Preparation Kit during the creation of a library from a DNA sample (e.g., genomics DNA from an organism of interest). The library consists of a set of single-stranded template DNA fragments representing the entire span of the sample sequence. Each fragment is flanked by suitable amplification and sequencing DNA adaptors (MID adaptors in this case), then purified and quantitated.

MIDs become part of the sequence of each read in the library, such that they provide a recognizable sequence tag at the beginning of each read. When multiple libraries are prepared with different MIDs, they can be sequenced together, in a single region of the PTP device; the data analysis software then deconvolutes the data and assigns each read to the correct library. Multiplex sequencing can greatly improve the utilization of available wells on the PTP device during a sequencing run. This technique can be especially useful for sequencing many small samples in parallel, such as samples from viruses, BACs, plasmids, small bacterial genomes, disease-associated regions, etc.