Genomics Services

Next-Generation Sequencing (NGS).

Next-generation sequencing (NGS), is a methodology that increases sequencing throughput by laying millions of DNA fragments on a single chip and sequencing all fragments in parallel. DNA fragments, which can be collected from various upstream processes, are used to build libraries that are then used as sequencing templates. These are prepared for sequencing by ligating specific adaptor oligonucleotides to both ends of each fragment. Following sequencing, informatics allows each sequencing read to be mapped to a reference genome. NGS platforms can be used for transcriptome profiling, miRNA profiling, DNA-protein interaction studies using chromatin immunoprecipitation (ChIP), and DNA methylation studies.

The Strengths and Weaknesses of NGS and Microarrays.

Microarray Analysis
Massively parallel, or next-generation, sequencing
Pros
Cons
Pros
Cons
Relatively inexpensive High background, low sensitivity Low background, very sensitive Expensive
Easy sample preparation Limited dynamic range Large dynamic range Complex sample preparation
Mature informatics and statistics Not quantitative Quantitative Limited bioinformatics
  Competitive hybridisation Massive information technology infrastructure required
Annotation of probes

(This table was taken from Asmann et al. Gastroenterology 2008, 135:1466)

Available NGS Technology Platforms.

Three commercially available NGS platforms, include Roche (454) FLX Genome Sequencer, Illumina Genome Analyzer II, and Applied Biosystems’ SOLiDTM (Sequencing by Oligo Ligation and Detection) System. In addition, single molecule sequencers, “third-generation sequencers”, have been developed. Helicos’ HeliScope, which employs True Single Molecule Sequencing (tSMS) technology to sequence samples without amplification, is capable of producing over 10 Gb of sequence data per 8 day run. Pacific Biosciences has developed Single Molecule Real Time (SMRTTM) sequencing technology which involves proprietary surface and nucleotide chemistries. This sequencer, which promises longer reads, shorter run times, and higher quality data, is expected to be released in 2010.

NGS Sequencing Chemistry.

Roche-Pyrosequencing involves the use of a pyrophosphate molecule, released following nucleotide incorporation by DNA polymerase, to propagate reactions that ultimately produce light.

Illumina- sequencing-by-synthesis involves the use of four differently labeled fluorescent nucleotides that have their 3’-OH groups chemically inactivated to ensure only a single base is incorporated per cycle. Each base incorporation cycle is followed by an imaging step to identify the base incorporated, and a chemical step that removes the fluorescent group and deblocks the 3’ end for the next base incorporation cycle.

Applied Biosystems/Life Technologies.

The SOLiD system uses a ligation-based sequencing process that starts by annealing a universal sequencing primer that is complementary to the SOLiD-specific adaptors on the library fragments. Then, a limited set of semi-degenerate 8-mer oligonucleotides (and DNA ligase) is added. When matching 8-mers hybridise to DNA fragment sequences adjacent to the universal primer, DNA ligase seals the phosphate backbone and a fluorescent readout identifies the fixed base of the 8- mer. A subsequent cleavage step removes bases 6-8 of the ligated 8-mer, removing the fluorescent group and enabling another round of 8-mer ligation, and so on. The advantage of ligation-based sequencing chemistry is the “built-in” quality check of read accuracy.

Amplification Approach.

Emulsion PCR is the amplification approach used by both Roche and Applied Biosystems. For the Roche platform, emulsion PCR is carried out on the DNA fragments attached to the surfaces of agarose beads. On the Applied Biosystems sequencer, the DNA fragements are attached to the surfaces of magnetic beads. Bridge amplification (Illumina) is a PCR reaction that occurs within a discrete area of the flow cell surface.

Read Length/Mb Per Run/Time Per Run/Approximate Cost.

NGS produces shorter reads (25-400 bp) with varying “read length” among different NGS platforms. Roche provides the longest read (400bp with the Titanium system) while Illumina and Applied Biosystems are much shorter (32-75 bp and 35 bp, respectively). The advantageous read length and run time of the Roche platform is offset by its cost (the Roche GS FLX sequencer’s “cost-per-run” is more expensive than the other two). Additionally, the FLX has lower run yield (roughly between 20% and 46% of the sequence data that could be obtained by using the Applied Biosystems and Illumina systems).

Comparison of Roche, Illumina, and Applied Biosystems NGS Platforms.

Roche (454): Titanium series reagents (run on FLX Genome Sequencer)
Illumina: Genome Analyser II
Applied Biosystems: SOLiDTM
Sequencing chemistry pyrosequencing polymerase-based sequencing-by-synthesis Ligation-based sequencing
Amplification approach Emulsion PCR Bridge amplification Emulsion PCR
MB/run 400-600 MB 1300 MB 3000 MB
Time/run 10 hr 4 days 5 days
Read length 400 bp up to 75 bp 35 bp

We are currently offering an “end to end” services solution for NGS at DNAmicroarray, Inc:

  1. Let us know details of your project in the form below.
  2. Specify the NGS technology platform of your choice.
  3. Our expert technical staff will outline a list of services that will best suit your project generating desired genomic analyses at the lowest cost.
  4. You send us your samples, cells, tissues, or RNA samples.
  5. Our certified staff and laboratories will perform the assays.
  6. We will send you back the analyzed data.

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