NGS Sequencing

Next Generation Sequencing Introduction

Features

  • Rich experience on sample treatments - SANGON has more than 10 years' experience in nucleic acid purification and sample preparation.
  • Comprehensive experiment platforms - SANGON has 454, Illumina, Ion Torrent sequencing and Sanger sequencing platforms. We could provide optimal solutions with flexible use of these platforms according to the research purpose or the requirement of experiments.
  • Cost-effective
  • Powerful Bioinformatics analysis and Follow-up Services - Bioinformatic teams could provide customized bioinformatics analysis services, to solve any problems from customers.
  • Abundant products and biological technology services - Sangon has molecular biology products more than 5000 and complete service system of biological technology. Based on the NGS service results, customers can choose other services in subsequent experiments.

Overview of Next Generation Sequencing (NGS)

High-throughput sequencing technology-the new technology of sequencing (Next Generation Sequencing), can parallelly sequence millions of DNA molecules. Although utilized quite diverse techniques and biochemistry in each step from template library preparation, fragment amplification, to sequencing, they all adopted a massive matrix configuration popularized by microarray analysis: DNA samples on the array are simultaneously analyzed in parallel. First, massive parallelism can be achieved through ordered or disordered array configuration that offers high degree of information density. This dramatically increases the overall throughput of the sequencing operation. Second, no electrophoresis steps is required. So it's widely used in Systems Biology, Function Genomics, Epigenomics, exon sequencing ,transcriptome, etc.

The representatives of NGS platforms are Roche 454, Illumina Solexa and Ion Torrent. These platforms could produce very large data per run, ranging from 0.5 ~600 G data, >100 bases in single read. The 454 Sequencer utilizes emulsion PCR to yield amplicons used for the sequencing procedure. Proportional amount of primers coated beads and library fragments are mixed together. The aqueous solution is mixed with oil to form emulsion. After PCR, each bead is coated with thousands of copies of DNA of the same sequence. Beads are further enriched, transferred, and deposited on a picotiter plate fabricated in organized array of tiny wells with each hole occupied by only one bead. The pyosequencing, as often called, relies on ATP sulfurylase and luciferase. Release of pyrophosphate, during nucleotide triphosphate incorporation into the DNA chain, triggers a cascade of biochemical reaction via ATP sulfurylase and luciferase, resulting in a burst of biochemiluminescent light being emitted. Sequencing is achieved by sequentially introducing each of the four dNTPs into the flow cell. Presence or absence of light burst of each picotiter well indicates the incorporation or not of corresponding nucleotide and, therefore, reveals the identity of complementary base on the template DNA in that well. Major advantages of pyrosequencing are its speed and read length-up to 500 bp.

Illumina's amplification of singlestranded library fragments is carried out through a process coined "bridge amplification" On an oligo-derived flow-cell surface, consisting eight independent lanes, single-strand DNAs flanked by asymmetrical adaptors form an oligo-bridges from both ends. After multiple PCR thermal cycles, thousands of copies of DNA, amplicons, based on one single-strand of DNA fragment are created and clustered on the surface to a single physical location. Illumina utilizes sequencing-by-synthesis with fluorescently labeled nucleotides and reversible terminators. In each cycle of sequence interrogation, four distinctly labeled nucleotides are added simultaneously to the flow cell channel to make DNA chain extension. Each nucleotide is 3'-OH blocked to prevent further addition. Then fluorescence images are acquired. The 3' end is then unblocked to allow next cycle of extension to occur. This process repeats multiple times, up to 2x100 cycles.

The sequence composition produced by Ion Torrent is determined by measuring pH changes due to hydrogen ion liberation as nucleotides are incorporated during strand synthesis in picolitre wells. The Ion-Chip has millions of ISFET-microwell.A microwell containing a template-DNA-coated bead(produced by emulsion PCR) to be sequenced is flooded with a single species of dNTP. If the introduced dNTP is complementary to the template, it is incorporated into the growing strand. This causes the release of a hydrogen ion that triggers an ISFET ion sensor, which indicates that a reaction has occurred. If homopolymer repeats are present in the template sequence, multiple dNTP molecules will be incorporated in a single cycle.

This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal. Using integrated circuits to measure pH changes to identify base incorporation removes the need for expensive light detection systems, substantially reduces costs and, theoretically, is in fiand a scalable; since the number of sequences obtained simply equates to the physical dimensions of the integrated ISFET sensor (Ion-Chip).

The 3rd NGS technologies, based on real-time single molecule sequencing, is rapidly being developed, but not yet for large-scale commercial application.

NGS Services at Sangon Biotech

SANGON has all three of those sequencing platforms mentioned above. With those platforms, SANGON has the capability to meet almost all of your NGS project requirement. Major applications are:

a.Genome sequencing:
  • de novo genome sequencing: de novo sequencing of unknown genome; annotation and functional analysis of the genome and gene sequences;
  • re-sequencing of the known genome; analysis of genome variation, such as CNV, SNP and chromosome structure variation, etc.

Metagenomic sequencing: 16S rDNA or ITS sequencing of all microorganisms in the same environment; revealing the diversity of microbial community, population structure, evolution, functional activity, mutual cooperation and relationships with the environment.

b.Exome Capture Sequencing:

Through the exon capturing technology to sequence the captured exon regions. Mutations/SNP in genes could be detected. Suitable for sequencing exon of cancer or Mendel disease.

c.Transcriptome sequencing:

Whole transcriptome sequencing: sequencing total transcribed mRNA in the sample;

Analysis: UTRs regions; new transcript analysis & annotation, RPKM calculation, DEG analysis, GO analysis, KEGG analysis, SNV/SNP, alternative splicing; identification of fusion genes, etc;

d.Small RNA sequencing:

Using total miRNA sequencing, you can profile the differential expression of known microRNAs as well as detect novel microRNA targets and wide-ranging sequence variation or "iso-miRs" miRBase accessions.

e.Other NGS service:

Chip-seq, Methylation-seq, whole genome genetic marker sequencing,etc.

More Information

How To Order

Download NGS Sequencing Order Form and email to NGS@sangon.com and copy orders to order@sangon.com


Frequently Asked Questions and Answers

Q: How much money does one NGS project need?

A: Different sequencing projects, sequencing platform, data and analysis are the factors that affect the price. In general, we assessed according to customer's requirements.

Q: How long to complete the NGS projects?

A: About three months, containing the bioinformatics' analysis.

Q: How much data do I need for the transcriptome sequencing?

A: The vast majority of the transcriptome projects are sequenced by Hiseq2000 platform. For the transcriptome sequencing, the more sequencing we have, the better the results will be. At present, we provides 5G raw data for each sample.

Q: I want to do 16 s/18s/ITS sequencing, how many samples does one 454 run can hold? How much data for each sample is enough?

A: We can get 800000-1000000 reads from each 454 run. The reads of samples should be decided by the clientele.