Illumina下一代测序系统MiSeqDx获加拿大监管部门批准
11:17原文出处：
　　Illumina公司表示，门已经同意批准该公司的MiSeqDx仪器，同时还准许该公司利用MiSeqDx进行囊性纤维化相关的139个突变基因的检测以及囊性纤维化的临床测序分析。　　根据该公司的说法，这是加拿大第一次批准下一代测序系统。　　Illumina公司在2013年获得了美国食品和药物针对该仪器和检测的上市前批准。同时，其MiSeqDx通用也获得了FDA的批准，包括试剂制备库、样品引物和可以用于研发自己的，基于MiSeqDx系统的实验的测序耗材。　　MiSeqDx囊性纤维化139突变检测实验能够同时检测139个临床相关的致病突变，这些突变主要存在于囊性纤维化跨膜调节基因。Illumina公司说，该检测涵盖了医学协会和加拿大大学医学遗传学家们建议需要筛查的所有的囊性纤维化突变类型，以及许多在其他族群中存在的致病突变。
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Cluster Generation and Sequencing
MiSeq Reagent Kit v2
Read Length
Total Time*
1 × 36 bp
540-610 Mb
2 × 25 bp
750-850 Mb
2 × 150 bp
4.5-5.1 Gb
2 × 250 bp
7.5-8.5 Gb
MiSeq Reagent Kit v3
Read Length
Total Time*
2 × 75 bp
3.3-3.8 Gb
2 × 300 bp
13.2-15 Gb
Reads Passing Filter+
MiSeq Reagent Kit v2
Single Reads
Paired-End Reads
MiSeq Reagent Kit v3
Single Reads
Paired-End Reads
Quality Scores++
MiSeq Reagent Kit v2
& 90% bases higher than Q30 at 1 x 36 bp
& 90% bases higher than Q30 at 2 x 25 bp
& 80% bases higher than Q30 at 2 x 150 bp
& 75% bases higher than Q30 at 2 x 250 bp
MiSeq Reagent Kit v3
& 85% bases higher than Q30 at 2 x 75 bp
& 70% bases higher than Q30 at 2 x 300 bp您的位置： >
来源：　　作者：周林文;
MiSeq:新一代个人化测序仪　 近年来ILLuMxNA公司先后推出了Hiseq和Miseq高性能测序仪，而后者更是新一代小型化的“椅上型”(bench一toP)测序仪的代表，备受期待。Miseq因其卓越的性能和小型化的优势，潜在的应用领域十分广泛，已不在局限于传统的生物实验室。下面是本刊记者就Miseq的相关话题对x LLuMINA亚太区总裁Tim orpin做了专访。((生物技术世界)):据我们所知，Mise口和Hiseq墓本上用的是相同的测序方法，那么与Hjseq相比，Miseq做了哪些改进，使其在小型化的同时来保证测序性能? Timo印in:Hiseq和Miseq的确有许多相同之处，它们所使用的技术很多是一样的。不过它们之间也有很多不同点，从一开始，Miseq就是以新一代个人化测序仪的定位来设计的。这是第一台真正让新一代测序技术走进每个人、每个实验室的测序仪。Hiseq的设计理念是追求最高的通量，尽可能读出更多的DNA序列。而Miseq的设计意图却有所不同，它追求的是效率:它可以在一个8小时的工作日内，从DNA开始分析一个样本，并获得最终结果。在这样的短时间内就完成了一个完整的工作流程。之所以能做到这点，部分是(本文共计3页)　　　　　　　　　　
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　　　　　　From Wikipedia, the free encyclopedia
Massive parallel sequencing or massively parallel sequencing is any of several high-throughput approaches to
using the co it is also called
(NGS) or second-generation sequencing. Some of these technologies emerged in
and became commercially available since 2005. These technologies use miniaturized and parallelized platforms for sequencing of 1 million to 43 billion short reads (50-400 bases each) per instrument run.
Many NGS platforms differ in engineering configurations and sequencing chemistry. They share the technical paradigm of massive parallel sequencing via spatially separated, clonally amplified
templates or single DNA molecules in a . This design is very different from that of —also known as capillary sequencing or first-generation sequencing—that is based on
separation of chain-termination products produced in individual sequencing reactions.
DNA sequencing with commercially available NGS platforms is generally conducted with the following steps. First, DNA sequencing libraries are generated by clonal amplification by
. Second, the DNA is sequenced by , such that the DNA sequence is determined by the addition of
to the complementary strand rather through chain-termination chemistry. Third, the spatially segregated, amplified DNA templates are sequenced simultaneously in a massively parallel fashion without the requirement for a physical separation step. While these steps are followed in most NGS platforms, each utilizes a different strategy.
NGS parallelization of the sequencing reactions generates hundreds of megabases to gigabases of nucleotide sequence reads in a single instrument run. This has enabled a drastic increase in available sequence data and fundamentally changed genome sequencing approaches in the biomedical sciences. Newly emerging NGS technologies and instruments have further contributed to a significant decrease in the cost of sequencing nearing the mark of $1000 per .
As of 2014, massively parallel sequencing platforms commercially available and their features are summarized in the table. As the pace of NGS technologies is advancing rapidly, technical specifications and pricing are in flux.
HiSeq 2000 sequencing machine
NGS Platforms
Template Preparation
Max Read length (bases)
Run Times (days)
Max Gb per Run
Clonal-emPCR
Pyrosequencing
GS FLX Titanium
Clonal-emPCR
Pyrosequencing
Illumina MiSeq
Clonal Bridge Amplification
Reversible Dye Terminator
Illumina HiSeq
Clonal Bridge Amplification
Reversible Dye Terminator
Illumina Genome Analyzer IIX
Clonal Bridge Amplification
Reversible Dye Terminator
Life Technologies SOLiD4
Clonal-emPCR
Oligonucleotide 8-mer Chained Ligation
Life Technologies Ion Proton
Clonal-emPCR
Native dNTPs, proton detection
Complete Genomics
Gridded DNA-nanoballs
Oligonucleotide 9-mer Unchained Ligation
Helicos Biosciences Heliscope
Single Molecule
Reversible Dye Terminator
Pacific Biosciences SMRT
Single Molecule
Phospholinked Fluorescent Nucleotides
10,000 (); 30,000+ (max)
Run times and gigabase (Gb) output per run for single-end sequencing are noted. Run times and outputs approximately double when performing paired-end sequencing. ?Average read lengths for the Roche 454 and Helicos Biosciences platforms.
Two methods are used in preparing templates for NGS reactions: amplified templates originating from single DNA molecules, and single DNA molecule templates. For imaging systems which cannot detect single fluorescence events, amplification of DNA templates is required. The three most common amplification methods are emulsion PCR (emPCR), rolling circle and solid-phase amplification. The final distribution of templates can be spatially random or on a grid.
methods, a
is first generated through random fragmentation of genomic DNA. Single-stranded DNA fragments (templates) are attached to the surface of beads with adaptors or linkers, and one bead is attached to a single DNA fragment from the DNA library. The surface of the beads contains
probes with sequences that are complementary to the adaptors binding the DNA fragments. The beads are then compartmentalized into water-oil emulsion droplets. In the aqueous water-oil emulsion, each of the droplets capturing one bead is a PCR
that produces amplified copies of the single DNA template.
Amplification of a population of single DNA molecules by
in solution is followed by capture on a grid of spots sized to be smaller than the DNAs to be immobilized.
Forward and reverse primers are covalently attached at high-density to the slide in a flow cell. The ratio of the primers to the template on the support defines the surface density of the amplified clusters. The flow cell is exposed to reagents for -based extension, and priming occurs as the free/distal end of a ligated fragment "bridges" to a complementary
on the surface. Repeated
and extension results in localized amplification of DNA fragments in millions of separate locations across the flow cell surface. Solid-phase amplification produces 100–200 million spatially separated template clusters, providing free ends to which a universal sequencing primer is then hybridized to initiate the sequencing reaction. This technology was filed for a patent in 1997 from Glaxo-Welcome's Geneva Biomedical Research Institute (GBRI), by Pascal Mayer, Eric Kawashima, and Laurent Farinelli, and was publicly presented for the first time in 1998. In 1994 Adams and Kron filed a patent on a similar, but non-clonal, surface amplification method, named “bridge amplification” adapted for clonal amplification in 1997 by Church and Mitra.
Protocols requiring DNA amplification are often cumbersome to implement and may introduce sequencing errors. The preparation of single-molecule templates is more straightforward and does not require PCR, which can introduce errors in the amplified templates. AT-rich and GC-rich target sequences often show amplification bias, which results in their underrepresentation in genome alignments and assemblies. Single molecule templates are usually immobilized on solid supports using one of at least three different approaches. In the first approach, spatially distributed individual primer molecules are covalently attached to the solid support. The template, which is prepared by randomly fragmenting the starting material into small sizes (for example,~200–250 bp) and adding common adapters to the fragment ends, is then hybridized to the immobilized primer. In the second approach, spatially distributed single-molecule templates are covalently attached to the solid support by priming and extending single-stranded, single-molecule templates from immobilized primers. A common primer is then hybridized to the template. In either approach, DNA polymerase can bind to the immobilized primed template configuration to initiate the NGS reaction. Both of the above approaches are used by Helicos BioSciences. In a third approach, spatially distributed single polymerase molecules are attached to the solid support, to which a primed template molecule is bound. This approach is used by Pacific Biosciences. Larger DNA molecules (up to tens of thousands of base pairs) can be used with this technique and, unlike the first two approaches, the third approach can be used with real-time methods, resulting in potentially longer read lengths.
and his student
at the Royal Institute of Technology in
published their method of . Pyrosequencing is a non-electrophoretic, bioluminescence method that measures the release of inorganic
by proportionally converting it into visible light using a series of enzymatic reactions. Unlike other sequencing approaches that use modified nucleotides to terminate DNA synthesis, the pyrosequencing method manipulates DNA polymerase by the single addition of a
in limiting amounts. Upon incorporation of the complementary dNTP, DNA polymerase extends the primer and pauses. DNA synthesis is reinitiated following the addition of the next complementary dNTP in the dispensing cycle. The order and intensity of the light peaks are recorded as flowgrams, which reveal the underlying DNA sequence.
This approach uses reversible terminator-bound dNTPs in a cyclic method that comprises nucleotide incorporation, fluorescence imaging and cleavage. A fluorescently-labeled terminator is imaged as each dNTP is added and then cleaved to allow incorporation of the next base. These nucleotides are chemically blocked such that each incorporation is a unique event. An imaging step follows each base incorporation step, then the blocked group is chemically removed to prepare each strand for the next incorporation by DNA polymerase. This series of steps continues for a specific number of cycles, as determined by user-defined instrument settings. The 3' blocking groups were originally conceived as either enzymatic or chemical reversal The chemical method has been the basis for the Solexa and Illumina machines. Sequencing by reversible terminator chemistry can be a four-colour cycle such as used by Illumina/Solexa, or a one-colour cycle such as used by Helicos BioSciences. Helicos BioSciences used “virtual Terminators”, which are unblocked terminators with a second nucleoside analogue that acts as an inhibitor. These terminators have the appropriate modifications for terminating or inhibiting groups so that DNA synthesis is terminated after a single base addition.
In this approach, the sequence extension reaction is not carried out by polymerases but rather by DNA
and either one-base-encoded probes or two-base-encoded probes. In its simplest form, a fluorescently labelled probe hybridizes to its complementary sequence adjacent to the primed template. DNA ligase is then added to join the dye-labelled probe to the primer. Non-ligated probes are washed away, followed by
to determine the identity of the ligated probe. The cycle can be repeated either by using cleavable probes to remove the fluorescent dye and regenerate a 5′-PO4 group for subsequent ligation cycles (chained ligation) or by removing and hybridizing a new primer to the template (unchained ligation).
Pacific Biosciences is currently leading this method. The method of real-time sequencing involves imaging the continuous incorporation of dye-labelled nucleotides during DNA synthesis: single DNA polymerase molecules are attached to the bottom surface of individual zero-mode waveguide detectors (Zmw detectors) that can obtain sequence information while
nucleotides are being incorporated into the growing primer strand. Pacific Biosciences uses a unique DNA polymerase which better incorporates phospholinked nucleotides and enables the resequencing of closed circular templates. While single-read accuracy is 87%, consensus accuracy has been demonstrated at 99.999% with multi-kilobase read lengths.
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: Hidden categories:&& 查看话题
二代高通量测序哪个平台好
miseq；454；ABI哪个平台性价比好。
454读长是400-500bp，但是较贵，不实用，现在测序主要是hiseq，还有就是算2.5代测序的ion torrent平台 现在国外的文献中用454测序的还是比较多的，再就是hiseq2000，,454读长要比hiseq长，个人觉得454出来的结果好处理一些，拼接结果更可信一些 454破产了,现在主要就是MiSeq : Originally posted by kiruwa at
454破产了,现在主要就是MiSeq Miseq 300*2双端测序在读长上有所进步，但是有关环境微生物群落直接测序大多都用454，不知道Miseq在发表论文时认可度如何？ : Originally posted by fcheng025 at
Miseq 300*2双端测序在读长上有所进步，但是有关环境微生物群落直接测序大多都用454，不知道Miseq在发表论文时认可度如何？... 没问题 都是常规方法了 本人目前做的测定方法就是Miseq 。。 : Originally posted by duzhize at
本人目前做的测定方法就是Miseq 。。 结果怎么样？委托哪里做的？ 用第三代吧！速度快，量大 : Originally posted by
用第三代吧！速度快，量大 太贵了呀}