Comparison of the nine sequencing platforms

Sanger

Enterprise: Life Technology

Launch time: invented in 1977, the first commercial sequencer in 1986

Mainstream model: 3730XL

Sample requirements: PCR product: concentration ≥ 50 ng / ul volume ≥ 10ul; plasmid: content ≥ 5ug; bacterial solution: volume ≥ 1ml.

Sequencing principle:

The dideoxy chain termination method: The principle of Sanger sequencing is that each reaction contains all four deoxynucleotide triphosphates (dNTPs) to be amplified and mixed with a limited amount of a different dideoxynucleoside triphosphate (ddNTP). ) to terminate it. Since the ddNTP lacks the 3'-OH group required for extension, the extended oligonucleotide is selectively terminated at G, A, T or C, the termination point being determined by the corresponding dideoxy in the reaction. The relative concentration of each of the dNTPs and ddNTPs can be adjusted so that the reaction yields a series of fragments ranging from a few to a few thousand apart, one base apart. They have a common starting point, but terminate in different nucleotides. High-resolution denaturing gel electrophoresis can be used to separate fragments of different sizes. After gel treatment, X-ray film can be autoradiographed or non-isotopically labeled. Detection.

Library preparation: no need to build a library, provide plasmids, bacterial liquids, PCR products, etc.

Template preparation: PCR amplification

Reading length: 500-1000bp

Sequencing flux:

Daily flux of four sequencing methods:

Short fragment sequencing 36cm36min500bp 1,728,000bp/day;

Rapid sequencing 36cm60min700bp 1,440,000bp/day;

Standard sequencing 36cm90min800bp 1,113,000bp/day;

The long fragment was sequenced at 50 cm 120 min 1100 bp 1,002,000 bp/day.

(36cm refers to capillary length, 36min refers to one reaction time, and 500bp refers to the read length of one reaction;)

Time / run: 36 min-2 hours

Base accuracy: 99.999%

Mutation detection ability (DNA resequencing aspect):

Heterozygous insertions and deletions,microsatellite, SNP, AFLP, T-RFLP, and LOH analyses

AFLP: Amplified Fragment Length Polymorphism,;

T-RFLP: terminal restriction fragment length polymorphism;

LOH analyses: loss of heterozygosity (Loss of Heterozygosity);

Cost: $95,000 per machine, about $4 per reaction, 800bp per reaction,

Data format: *.bcf ; *.abl

Analyzing Software:

Data Collection Software Data Collection v2.0 (available)

Manages your instrument setup, controls instrument operations, allows real-time data visualization, and performs diagnostics. New features include:

- Auto-analysis with GeneMapper® v3.5 and SeqScape® v2.1

- FDA 21CFRpart 11 compliance (for US customers)

- Flexibility to use any choice in dye set option

- Additional optimized sequencing run modules covering more applications

Sequence Analysis Software Sequencing Analysis v5.1

Designed to base-call; assign quality values; trim, display, edit and print DNA sequencing data using the KB basecaller

Sequence splicing and comparative gene analysis software Seqscape® v2.1

Provides everything you need to perform resequencing applications such as VariantSEQrTM Resequencing System

Automated gene fragment analysis software GeneMapper® v3.5

Enables configurable, automated allele calling -- a plus for high-throughput genotyping and includes tools for SNPlexTM data analysis

SNP classification data management software BioTrekkerTM v1.0 (optional data-mining tool)

Provides a fast, powerful way to search for, retrieve, and manage millions of genotypes

Advantages: Sequencing gold standard, read long

Disadvantages: low throughput and high cost

Classic case: Sanger sequencing belongs to the gold standard of sequencing technology, many articles

Remarks: Due to the limitations of the sequencing method itself, 10bp~40bp after sequencing primers cannot guarantee accuracy.

2.454

Business: Roche

Launch time: 2005

Mainstream models: Genome Sequencer FLX Titanium (launched in 2008)

Sample requirements: 5 μg of DNA, concentration ≥300 ng/μL

Sequencing principle: pyrosequencing

At the time of sequencing, a plate called "Pico TiterPlate" (PTP) was used, which contained more than 1.6 million pores composed of optical fibers containing various enzymes and substrates required for chemiluminescence reactions. At the beginning of sequencing, the four bases placed in four separate reagent bottles are sequentially circulated into the PTP plate in the order of T, A, C, and G, entering only one base at a time. If base pairing occurs, a pyrophosphate is released. This pyrophosphate is subjected to a synthesis reaction and a chemiluminescence reaction under the action of various enzymes, and finally fluorescein is oxidized to oxyluciferin, and an optical signal is released. The light signal emitted by this reaction is captured in real time by the instrument's configured high sensitivity CCD. When one base is paired with a sequencing template, one molecule of the optical signal is captured; thus, one-to-one correspondence can accurately and quickly determine the base sequence of the template to be tested.

Library preparation: Fragment (300-800 bp), Mate-pair

Template preparation: microemulsion PCR

Reading length: 400bp

Sequencing flux: 0.4-0.6 Gb/run

Time / run: 10 hours

Base accuracy: Q20 read length is 400 bases

Variability in detection (DNA resequencing: SNP, Indel, SV, CNV

Cost: $500,000 per unit, sequencing $5,600 per whole plate

Data format: Raw data: SFF format

Analysis software: GS De Novo Assembler software; GS Reference Mapper software; GS Amplicon Variant Analyzer software

Advantages: The outstanding advantage is that the reading is long, making the subsequent sequence stitching work more efficient and accurate. Fast, a sequencing reaction takes 10 hours and gets 400-600 million base pairs.

Disadvantages: The length of the homopolymer cannot be accurately measured, so the error rate for detecting the insertion deletion mutation is high; the flux is small and the cost is high.

Classic case: 1.Complete Khoisan and Bantu genomes from southern Africa.Schuster SC et al. (2010) Nature 463(7283): 943-7.

(See https:// ... ng/genome/index.jsp for more details)

Remarks: Particularly suitable for de novo splicing and metagenomic applications, mostly for new bacterial genomes. Too expensive for re-sequencing, not suitable

3.Solid

Enterprise: Life Technology (2008, ABI and Invitrogen merged into Life Technology)

Launch time: 2007

Mainstream model: SolidTM 4.0 (launched in 2010)

Sample requirements:

• Fragment Library 10 ng-5 μg

• Paired-end library 10 ng-5 μg

• Mate-paired library 5 μg-20 μg

Sequencing principle: sequencing while connecting

The sequencing primer and the linker can complement each other, and the 5' end can be ligated to an oligonucleotide complementary to the adjacent sequence. The octamer oligonucleotides can be competitively linked to the primers (the fourth and fifth positions of the oligomer are fluorescent marker sites). When the marker color is read, the oligonucleotide to be ligated is cleaved between the fifth and sixth positions to remove the marker, and the next round of reaction is performed, thereby sequentially circulating. In the first round of reactions, the determined base sites are: 4, 5, 9, 10, 14, 15 bases, and the like. Repeat the reaction process, shift one base, and use a primer that is one base less than the first round. The base sites that can be determined include: 3, 4, 8, 9, 13, 14, etc. Until offset to the first base of the primer (ie, base 0 of the sequence to be tested). Since the base of the site is known, the base type of the site 1 can be known by the fluorescence color of the read, and then the base type of the site 2 is inferred by the fluorescence color of the base 1 base, and so on. Until the entire sequence is read.

Library preparation:

• Mate-paired library (insert 600 bp to 10 Kb)

• Paired-end library

• Fragment Library

Template preparation: microemulsion PCR

Read length: 2*50 bp

Sequencing flux: 100-120 Gb/run, 12G/day

Time / run: 7~12 days

Base Accuracy: The accuracy of the original base data is greater than 99.94%, and the accuracy at 15X coverage depth can reach 99.999%.

Variability in detection (DNA resequencing): SNP, Indel, SV, CNV

Cost: $495,000 per unit, sequencing $6000/100Gb

Data format: Raw data: *csfasta and *quel; comparison output format: BAM/SAM.

Analysis software: Analysis software: Bioscope, there are many third-party software support for the format.

Advantages: High accuracy, 2 times per DNA base detection, increasing the accuracy of sequence reading

Disadvantages: Long running time, high error rate for detecting base substitution mutations

Classic case: SOLiD sequencing technology only published 60 articles in DNA applications, including 15 high-level articles published in Science/Nature and its sub-publishes, including 3 de nove sequencing articles, 26 targeted resequencing articles, whole 31 articles on genome resequencing. SOLID is equally widely used in articles such as RNA and Appearance.

Remarks: Industry-leading accuracy enables critical bio-variation detection for applications such as whole-genome resequencing, directed resequencing, and full transcriptome analysis.

4.HiSeq2000

Enterprise: Illumina

Launch time: 2010/1/1

Mainstream model: Illumina HiSeq2000

Sample requirements: 6ug/sample, no RNA, protein contamination, no degradation, OD260/OD280=1.8-2.0

Sequencing principle: sequencing while synthesizing

This sequencing technique attaches random fragments of genomic DNA to optically transparent surfaces that are extended by extensions and bridges to form Flowcells with hundreds of millions of clusters, each with approximately 1000 copies of the same DNA template. Then, four different fluorescently labeled bases whose ends are blocked are sequenced while synthesizing. This new approach ensures high accuracy and true sequencing of one base by base, eliminating specific errors in sequence and sequencing of homopolymers and repeats. This technology attach random fragment of genome DNA to optically transparent surface. And by extention bridge amplification of the DNA fragments, it create a flow cell with > 10 million clusters, each containing ~1,000 copies of same template. Then sequence by synthese using four kind Of terminal-closed base with different fluorescent mark. This technology assures the high accuracy and one by one base sequencing, avoids special mistakes of sequence and can be applied for repeated sequence and homopolymer.

Library preparation: Fragment, Mate-pair (200bp-40kb)

Template preparation: bridge amplification

Reading length: 50SE, 91PE, 101PE, etc.

Sequencing flux: 25G/day

Time / run:

91/101PE, 8-10 days;

50SE, 3 days;

150PE, 15 days;

In general, the strategy is different and the time is different.

Base accuracy: Q30% ≥ 80%; Q20% ≥ 90%

Variability in detection (in terms of DNA resequencing): SNP, Indel, SV, CNV

Cost: $690,000 per machine , $6000 per human genome sequencing(30X)

Data format: Sequencing results: *.fq; alignment: *. SOAP/*.BAM/*.SAM; variation: *.gff

Analysis software: GenomeStudio or third-party software package;

Advantages: large throughput, flexible sequencing, and diversified analysis software

Disadvantages: The cost is currently higher than the third generation sequencing, the sample preparation process is complex, and the sample requirements are relatively high.

Classic case: Initial genome sequencing and analysis of multiple myeloma. Chapman MA, et al. Nature. 2011 Mar 24;471 (7339): 467-72.

Cytoplasmic intron sequence-retaining transcripts can be dendritically targeted via ID element retrotransposons. Buckley PT, et al. Neuron. 2011 Mar 10;69(5):877-84.

Identification of fusion genes in breast cancer by paired-end RNA-sequencing. Edgren H, et al. Genome Biol. 2011 Jan 19;12(1):R6.

Genome-wide association mapping to candidate polymorphism resolution in the unsequenced barley genome. Cockram J, et al. Proc Natl Acad Sci US A. 2010 Dec 14;107(50):21611-6. Epub 2010 Nov 29.

MHC class II transactivator CIITA is a recurrent gene fusion partner in lymphoid cancers. Steidl C, et al. Nature. 2011 Mar 17;471(7338):377-81. Epub 2011 Mar 2.

Tumour evolution inferred by single-cell sequencing.Navin N, et al. Nature. 2011 Apr 7;472(7341):90-4. Epub 2011 Mar 13.

Remarks: The additional benefit is experimental flexibility-application that require different read lengths can be run on each flowcell – an example is that a whole genome sequencing and de novo sequencing Study (eg. Of human and microorganism) could be run on one flowcell at 2 x 100bp reads, and the other could be running ChIP-seq and gene expression samples at 1 x 50bp read length. Each flowcell is controlled independently; Run can be started/stopped independent of the other.

5.Helicos

Enterprise: Helicos Biosciences

Launch time: 2008

Mainstream model: HeliScopeTM Single Molecule Sequencer (launched in 2008)

Sample requirements: 1-3 μg, starting DNA volume does not exceed 100 μL.

Sequencing principle: sequencing while synthesizing, reversible blocking sequencing

The DNA to be tested was randomly broken into small fragments, poly-dA was added to the end of each small fragment (~200 bp), and multiple poly-dT primers were randomly immobilized on the glass chip, and the ends were fluorescently labeled. To facilitate precise positioning. First, the small fragment DNA template was hybridized with the poly-dT primer on the detection chip and precisely positioned, and then the fluorescently labeled end terminator was added one by one. This terminator is not the same as the terminator of Illumina, it is not four-color, it is monochromatic, meaning that all terminators are labeled with the same dye. After incorporation of a single fluorescently labeled nucleotide, washing, monochromatic imaging, followed by cleavage of the fluorescent dye and inhibitory group, washing, capping, allowing for the incorporation of the next nucleotide. A large number of sequences can be read in real time through repeated cycles of incorporation, detection, and ablation. Finally, with the aid of the software system, the complete nucleic acid sequence can be analyzed.

Library preparation: Fragment, Mate-pair

Template preparation: single molecule detection

Read length: 25-55 bp, average 35 bp

Sequencing throughput: 21 to 35 Gb/run

Time / run: 8 days

Base accuracy: >20X coverage with more than 99.995% consistency

Variability in detection (in terms of DNA resequencing): SNP, CNV

Cost: Instrument $999,000/set, sequencing $0.45-0.60/Megabase.

Data format: Raw data: srf or sms format

Analysis software: It is recommended to use Helisphere open source software for filtering comparison

Advantages: True single-molecule sequencing, no pre-amplification, no biasing; especially suitable for RNA-Seq or direct RNA sequencing because it allows direct sequencing of RNA templates without the need to convert them to cDNA. The error rate for detecting base substitution mutations is very low, ~0.2%.

Disadvantages: high error rate, Insertion 1.5%, Deletion 3.0%; Heliscope will encounter some difficulties in the face of homopolymer, but can improve accuracy through secondary sequencing; due to the possible incorporation of unlabeled alkali in the synthesis Base, so its main source of error is missing.

Classic case: 1.Single-molecule sequencing of an individual human genome.

Dmitry Pushkarev, Norma F Neff & Stephen R Quake. Nat Biotechnol, 27. (9): 847-852.

(See http:// ... id/169/Default.aspx for more details)

Remarks: Particularly suitable for RNA-Seq or RNA direct sequencing applications

6.DNA Nanoball array

Enterprise: Pacific Biosciences

Launch time: Science paper published at the end of 2009, launched in 2010

Mainstream model: DNA Nanoball array

Sample Requirements: DNA by

PicoGreen Quantitative: 10ug recommended, at least 7.5ug. Concentration: 75-150 ng / ul; volume: 50-200 ul; DNA length: large molecular weight double-stranded genomic DNA, most of which exceeds 20 kb.

Sequencing principle: sequencing while connecting

High-density DNA (glass plate) nanochip technology and non-continuous, non-linked, probe-anchor-ligation (cPAL) technology were used for sequencing. The genome was randomly broken into 500 bp random length fragments, ligated at both ends, ligated, restriction endonuclease digestion, repeated 2 times, and finally ligated into a DNA loop. At present, the 4-link construction method can support 70 bp single End sequencing (35 bp double-end sequencing). Next, as shown, each DNA loop was amplified in the reaction solution at a high speed to form a nanosphere (DNB), so that each DNA loop was amplified approximately 200 times. These nanospheres are then tiled onto a pre-treated glass plate to form a nanochip. Finally, sequencing was performed by a non-linkage-linked probe-anchor ligation (cPAL) technique. The 10 bp probe has an anchor base (A or T or G or C), and all other positions are N, which hybridizes to the template. The ligation reaction has four different fluorescent signals depending on the four different bases (A/T/G/C), requiring a total of 40 different anchor probes. Each anchor probe is eluted after hybridization. Through the fluorescence development results and decoding analysis of the DNB chip, we can determine the nucleic acid sequence of each DNB.

Library preparation: 500bp

Template preparation: Nanospheres (DNB)

Reading length: 35PE

Sequencing flux: 20-60G/run/flow slide, total 18 flow slides

Time / run: 9 days

Base accuracy: 99.999%

Variability in detection (in terms of DNA resequencing): SNP, Indel, SV, CNV

Cost: Only sequencing services are available, and instruments are not sold. The WGS cost for November 2009 was $1726 (45x), $8005 (87x)

Data format: Basic text format, *.tsv. Deliverables include: sequence variation, functional annotations, data summary reports, and supporting data for a full set of these results.

The data consists of three parts, the main part is reads and mappings, followed by assambly and variants, and the summary report is the least.

Analysis software: Genome comparison tools, Format conversion tools, Annotation tools, Reference tools, as well as tumor-normal sample comparison tools and tools for family genomic analysis, and large-scale comparisons for cancer genome analysis. Tools can simultaneously compare hundreds of genomes. Complete Genomics Assembly Pipeline version 1.5.0

Advantages: Automated sequencing, low cost, large throughput

Disadvantages: Length of reading, analysis software is not open, sample requirements are high

Classic case: Identification by whole-genome resequencing of gene defect responsible for severe hypercholesterolemia. Jonathan Rios, et al. Human Molecular Genetics, Volume19, Issue22 Pp. 4313-4318.

Human Genome Sequencing Using Unchained Base Reads on Self-Assembling DNA Nanoarrays. Radoje Drmanac1, et al. Science, Vol. 327 no. 5961 pp. 78-81 .

Analysis of Genetic Inheritance in a Family Quartet by Whole-Genome Sequencing. Jared C. Roach, et al. Science, Vol. 328 no. 5978 pp. 636-639

The mutation spectrum revealed by paired genome sequences from a lung cancer patient. William Lee, et al. Nature, Volume: 465, Pages: 473–477

Remarks: mapping reads: 40X/genome

7.The PacBio RS system

Enterprise: Pacific Biosciences

Launch time: Early trial started in February 2010

Mainstream model: PacBio RS

Sample requirements: 500 ng below 1 kb, purified genomic DNA, BACs, cDNA library or PCR product

Sequencing principle: sequencing while synthesizing, single molecule, real-time, or SMRTTM, technology

At the heart of this technology is the use of Zero-Mode Waveguide (ZMW) (Zero Band Boundary). There are tens of thousands of wells on the sequencing platform, and a single DNA polymerase and DNA strands to be sequenced are fixed in each well. Fluorescently labeled deoxynucleotides (A, T, C, G) were added to each well. Each deoxynucleotide can emit different wavelengths of fluorescence after activation. The bottom of the well has a very small hole that is as small as a single wavelength of the probe laser. According to our common sense, this hole is too small, and the laser cannot pass through the bottom of the well, thus failing to excite the fluorescent substance on the deoxynucleotide. Therefore, the microscope at the bottom detects a darkness. But we know that light is a wave that diffracts. Although the laser can't completely illuminate the whole well through the small hole, it can barely illuminate a small area near the small hole through the small hole. The DNA polymerase is just fixed in this small area. When a single deoxynucleotide is loaded on the DNA polymerase to form a new chemical bond, the fluorescent substance on the deoxynucleotide is activated to emit light, which is observed by a microscope. Fluorescence of this particular color lasts only a short period of time, and after the base is synthesized on the DNA strand, its fluorophore is cleaved off, continuing the synthesis of the next base. When the DNA strand synthesis is completed, it is also the time when the DNA strand sequencing is completed. However, the activity of DNA polymerase is gradually weakened under laser irradiation, and the synthesis reaction cannot be carried out in an infinite length, so the sequencing length of the DNA strand is also limited.

Library preparation: Fragment, Mate-pair (250bp-6kb)

Template preparation: SMRTbellTM library, no routine amplification required, only 500 ng samples are required for libraries below 1 kb

Reading length: 1000bp

Sequencing throughput: Highly flexible sequencing throughput

Time / run: Template preparation to primary basecall analysis takes less than a day, usually takes less than 4 hours.

Base Accuracy: The accuracy of social assessment is not high. The NEJM magazine published in January 2011 (on the sequencing of cholera in Haiti) mentioned its single base accuracy rate of only 81-83% in its supplementary.

The social evaluation of its accuracy rate is not good. Some websites say that its accuracy is very poor. The average error rate of single sequencing is 15%, and it is reduced to 8% after cycle sequencing.

Mutation detection ability (DNA resequencing): SNP, Indel, SV, CNV, can directly perform methylation and other modification sequencing, which is convenient for apparent research

Cost: $700,000 per machine, $99 per SMRTTMCell, each patterned with 15,000ZMW, each ZMW contain a single DNA polymerase.

Data format: bas.h5 – Base File and Filtered Bases File - Documentation

Cmp.h5 – Reference Mapped File and Consensus File - Documentation

Analysis software: mapping: BLASR (Basic Linear Alignment with Successive Refinement); assembly: ALLORA (A Long Read Assembler); SNP and Indel: RCCS (Reference Circular Consensus Sequencing) client software: SMRT Portal; provide: experiment specific, data- Rich reports in industry-standard output formats containing both primary and secondary analysis data. Third party software can be used.

Advantages: long reading; no need for PCR amplification, avoiding the resulting bias; less sample required; less time for sample preparation; remote data acquisition and sequencing parameters with RS system; flexible flux Fast time

Disadvantages: Low accuracy, requiring cycle sequencing,

Classic case: The Origin of the Haitian Cholera Outbreak StrainChen-Shan Chin, et al.N Engl J Med 2011; 364:33-42January 6, 2011.

Real-Time DNA Sequencing from Single Polymerase Molecules. John Eid, et al.Science 2 January 2009:

Vol. 323 no. 5910 pp. 133-138

DOI: 10.1126/science.1162986

Remarks: 1. After the sample is prepared, there is a run design step, which can be used on the client computer with RS remote software from the main selection mode and subsequent personalized analysis services, and the first time to receive sequencing data 2. As of 2011 In May, several RS sequencers were officially released, including the National Center for Biochemical Defense Analysis and Response (NBACC) and the Cold Spring Harbor Laboratory.

8.PGM

Enterprise: Ion Torrent

Launch time: 2010

Mainstream model: Ion Personal Genome MachineTM (2010)

Sample requirements: 5 μg DNA, starting DNA volume not exceeding 130 μL

Sequencing principle: semiconductor chip sequencing

The sequencer uses a high density semiconductor aperture chip. The chip is placed on an ion-sensitive layer and an ion susceptor. Whenever a nucleotide molecule is incorporated, protons are released, and the ion receptor senses the signal, knowing which nucleotide was Incorporation, thereby reading out the DNA sequence.

Library preparation: Fragment (185–210 bp)

Template preparation: PCR amplification

Reading length:

314 chip, 100 bp;

316 chip, 100bp;

318 chip, 200 bp.

Sequencing flux:

314 chip, 10 Mb/run;

316 chip, 100 Mb/run;

318 chip, 1Gb/run.

Time / run: 2 hours

Base accuracy: Consistency over 99.99%, >99.5% raw accuracy.

Variability in detection (in terms of DNA resequencing): SNP, Indel

Cost: $50,000 per unit, sequencing $500/run

Data format: Standard SFF or FASTAQ format

Analysis software: a series of commercial software for mutation detection, denovo assembly

Benefits: Unparalleled speed and 2 hours of sequencing; Ion Torrent's chemical sequencing principle is simple and natural, with no modified nucleotides, no lasers or optical detection equipment, resulting in minimal sequencing bias and excellent sequencing Cover the balance.

Disadvantages: The sequencing throughput is not large enough at present. Increasing the capacity of semiconductor chips is expected to improve the processing power of the sequencer.

Classic case: 1 After the outbreak of E. coli in Germany, Huada Gene used the third-generation sequencer, Ion Torrent, to perform genome-wide sequencing of the E. coli. Three days after the virus sample was obtained, BGI completed the genome sequencing of the new E. coli. After preliminary information analysis, the strain was found to be a new type of Escherichia coli with characteristics such as invasion, toxin production and intestinal bleeding.

Remarks: Particularly suitable for microbial genome sequencing and amplicon resequencing

9.MiSeq

Enterprise: Illumina

Launch time: 2011

Mainstream model: MiSeq Personal Sequencing System (2011)

Sample Requirements: Use Nextera, 50 ng DNA; use TruSeq, 100 ng–1 μg DNA

Sequencing principle: sequencing while synthesizing

This sequencing technique attaches random fragments of genomic DNA to optically transparent surfaces that are extended by extensions and bridges to form Flowcells with hundreds of millions of clusters, each with approximately 1000 copies of the same DNA template. Then, four different fluorescently labeled bases whose ends are blocked are sequenced while synthesizing. This new approach ensures high accuracy and true sequencing of one base by base, eliminating specific errors in sequence and sequencing of homopolymers and repeats.

Library preparation: Fragment

Template preparation: bridge amplification

Reading length:

1*35 bp

2*100 bp

2*150 bp

Sequencing flux:

1*35 bp >120 Mb/run

2*100 bp >680 Mb/run

2*150 bp >1 Gb/run

Time / run:

Total time for amplification and sequencing:

1*35 bp 4 hours

2*100 bp 19 hours

2*150 bp 27 hours

Base accuracy:

>90% bases higher than Q30 at 1*35 bp

>80% bases higher than Q30 at 2*100 bp

>75% bases higher than Q30 at 2*150 bp

Mutation detection ability (DNA resequencing aspect):

Cost: N/A

Data format: Instrument $125,000/set, sequencing $750/run

Analysis software: *.bcl, FASTQ, BAM, *.vcf and *.csv.

Advantages: Simple and fast sample preparation, sequencing and data processing on one instrument; reliable chemical method, reversible stop-base sequencing

Disadvantages: N/A

Classic case: N/A

Remarks: Particularly suitable for amplicon sequencing, cloning and minigenome sequencing

Source: Bio Abstracts

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