450:
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.
540:
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.
527:. All the key concepts of sequencing by synthesis were introduced, including (1) amplification of DNA to enhance the subsequent signal and attach the DNA to be sequenced (template) to a solid support, (2) generation of single stranded DNA on the solid support (3) incorporation of nucleotides using an engineered polymerase and (4) detection of the incorporated nucleotide by light detection in real-time. In a follow-up article, the concept was further developed and in 1998, an article was published in which the authors showed that non-incorporated nucleotides could be removed with a fourth enzyme (
1094:
112:
733:
1736:
704:
1755:
475:. An engineered polymerase is used to synthesize a copy of a single strand of DNA and the incorporation of each nucleotide is monitored. The principle of sequencing by synthesis was first described in 1993 with improvements published some years later. The key parts are highly similar for all embodiments of SBS and include (1)
1662:
759:
1855:
449:
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
99:
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
432:
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
569:
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
557:
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).
95:
to the complementary strand rather than 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. These steps are followed in most NGS platforms,
552:
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,
539:
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
479:
to enhance the subsequent signal and to attach the DNA to be sequenced to a solid support, (2) generation of single stranded DNA on the solid support, (3) incorporation of nucleotides using an engineered polymerase and (4) detection of the incorporation of nucleotide. Then steps 3-4 are
369:
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
574:
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
389:
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
53:. Some of these technologies emerged between 1993 and 1998 and have been commercially available since 2005. These technologies use miniaturized and parallelized platforms for sequencing of 1 million to 43 billion short reads (50 to 400 bases each) per instrument run.
1424:
419:
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
394:
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
359:
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.
107:
As of 2014, massively parallel sequencing platforms are 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.
1479:"Next generation sequencing for clinical diagnostics-principles and application to targeted resequencing for hypertrophic cardiomyopathy: a paper from the 2009 William Beaumont Hospital Symposium on Molecular Pathology"
699:, Adams CP, Kron SJ, "Method for performing amplification of nucleic acid with two primers bound to a single solid support", published 1997-06-24, assigned to Mosaic Technologies Inc. and
575:
demonstrated at 99.999% with multi-kilobase read lengths. In 2015, Pacific
Biosciences released a new sequencing instrument called the Sequel System, which increases capacity approximately 6.5-fold.
480:
repeated and the sequence is assembled from the signals obtained in step 4. This principle of sequencing-by-synthesis has been used for almost all massive parallel sequencing instruments, including
56:
Many NGS platforms differ in engineering configurations and sequencing chemistry. They share the technical paradigm of massive parallel sequencing via spatially separated, clonally amplified
1753:, Drmanac R, Callow MJ, Drmanac S, Hauser BK, Yeung G, "Single molecule arrays for genetic and chemical analysis", published 2013-05-21, assigned to Callida Genomics Inc.
441:
and Steve 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.
80:
DNA sequencing with commercially available NGS platforms is generally conducted with the following steps. First, DNA sequencing libraries are generated by clonal amplification by
1940:
Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, et al. (June 2013). "Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data".
370:
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.
1220:
2012:
1090:
1033:
1425:"Pacific Biosciences Introduces New Chemistry With Longer Read Lengths to Detect Novel Features in DNA Sequence and Advance Genome Studies of Large Organisms"
72:
separation of chain-termination products produced in individual sequencing reactions. This methodology allows sequencing to be completed on a larger scale.
1998:
1984:
433:
initiate the sequencing reaction. This technology was filed for a patent in 1997 from Glaxo-Welcome's Geneva
Biomedical Research Institute (GBRI), by
100:
emerging NGS technologies and instruments have further contributed to a significant decrease in the cost of sequencing nearing the mark of $ 1000 per
571:
1731:, Church GM, Porreca GJ, Shendure J, Rosenbaum AM, "Nanogrid rolling circle DNA sequencing", published 2017-04-18, assigned to
658:
Ronaghi M, Karamohamed S, Pettersson B, Uhlén M, Nyrén P (November 1996). "Real-time DNA sequencing using detection of pyrophosphate release".
1173:"Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding"
1089:, Ju J, Li Z, Edwards JR, Itagaki Y, "Massive parallel method for decoding DNA and RNA", published 2010-09-07, assigned to
1058:
700:
1894:
17:
1770:
1869:
1732:
1658:
1463:
1772:
A very large scale, high throughput and low cost DNA sequencing method based on a new 2-dimensional DNA auto-patterning process
1594:"Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations"
1224:
1657:, Church GM, Mitra R, "Replica amplification of nucleic acid arrays", published 2002-11-26, assigned to
1529:
Chee-Seng K, Yun LE, Yudi P, Kee-Seng C (April 2010). "Next
Generation Sequencing Technologies and Their Applications.".
513:
2020:
1037:
531:) allowing sequencing by synthesis to be performed without the need for washing away non-incorporated nucleotides.
2040:
584:
754:
725:
424:-based extension, and priming occurs as the free/distal end of a ligated fragment "bridges" to a complementary
438:
411:
in solution is followed by capture on a grid of spots sized to be smaller than the DNAs to be immobilized.
408:
594:
589:
429:
437:, Eric Kawashima, and Laurent Farinelli, and was publicly presented for the first time in 1998. In 1994
1111:
Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, Brown CG, et al. (November 2008).
548:
In this approach, the sequence extension reaction is not carried out by polymerases but rather by DNA
1985:"PacBio Users Report Progress in Long Reads for Plant Genome Assembly, Tricky Regions of Human Genome"
1847:
1750:
1728:
1654:
1086:
750:
721:
696:
1850:, Balasubramanian S, "Polynucleotide sequencing", published 2004-12-21, assigned to
1309:
Shendure J, Porreca GJ, Reppas NB, Lin X, McCutcheon JP, Rosenbaum AM, et al. (September 2005).
626:"Solid Phase DNA Minisequencing by an Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay"
382:
81:
512:
was first described in 1993 by combining a solid support with an engineered DNA polymerase lacking
1919:
1013:"2008 Release: NHGRI Seeks DNA Sequencing Technologies Fit for Routine Laboratory and Medical Use"
625:
1171:
McKernan KJ, Peckham HE, Costa GL, McLaughlin SF, Fu Y, Tsung EF, et al. (September 2009).
1775:. Fifth International Automation in Mapping and DNA Sequencing Conference. St. Louis, MO, USA.
1249:
Drmanac R, Sparks AB, Callow MJ, Halpern AL, Burns NL, Kermani BG, et al. (January 2010).
476:
463:
The objective for sequential sequencing by synthesis (SBS) is to determine the sequencing of a
1448:
1062:
1898:
816:"Massive parallel sequencing in forensics: advantages, issues, technicalities, and prospects"
1793:
1605:
1379:
1322:
1262:
1124:
554:
1681:"In situ localized amplification and contact replication of many individual DNA molecules"
728:), "Method of nucleic acid amplification", published 2007-06-13, assigned to
8:
1366:
Peters BA, Kermani BG, Sparks AB, Alferov O, Hong P, Alexeev A, et al. (July 2012).
566:
485:
329:
1873:
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1128:
1965:
1851:
1829:
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815:
729:
116:
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Mayer P, Matton G, Adessi C, Turcatti G, Mermod JJ, Kawashima E (October 7–10, 1998).
1705:
1680:
1628:
1593:
1251:"Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays"
1969:
1957:
1821:
1813:
1710:
1633:
1578:
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1508:
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1405:
1340:
1280:
1202:
1150:
986:
945:
896:
845:
796:
675:
481:
101:
68:—also known as capillary sequencing or first-generation sequencing—which is based on
65:
1833:
1352:
1292:
1949:
1805:
1749:
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1623:
1613:
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1132:
998:
976:
935:
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886:
876:
835:
827:
786:
667:
637:
1999:"PacBio Launches Higher-Throughput, Lower-Cost Single-Molecule Sequencing System"
1809:
791:
774:
425:
391:
88:
69:
561:
1727:
1598:
Proceedings of the
National Academy of Sciences of the United States of America
1494:
981:
964:
931:
831:
509:
493:
472:
61:
38:
1113:"Accurate whole human genome sequencing using reversible terminator chemistry"
2034:
1817:
497:
342:
1696:
1618:
1549:
Metzker ML (January 2010). "Sequencing technologies - the next generation".
1368:"Accurate whole-genome sequencing and haplotyping from 10 to 20 human cells"
1335:
1310:
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1961:
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92:
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1953:
916:"Massively parallel sequencing: the next big thing in genetic medicine"
524:
468:
421:
41:
using the concept of massively parallel processing; it is also called
1592:
Dressman D, Yan H, Traverso G, Kinzler KW, Vogelstein B (July 2003).
1311:"Accurate multiplex polony sequencing of an evolved bacterial genome"
1562:
543:
1110:
865:"Next generation DNA sequencing and the future of genomic medicine"
757:, "Method of nucleic acid sequencing", published 2004-06-23
720:
379:
84:
749:
1085:
1012:
657:
528:
521:
27:
DNA sequencing using the concept of massively parallel processing
775:"Next-generation sequencing: from basic research to diagnostics"
534:
549:
414:
1895:"True Single Molecule Sequencing (tSMS™): Helicos BioSciences"
1170:
91:, such that the DNA sequence is determined by the addition of
1059:"HiSeq v4 is here… and it delivers | Edinburgh Genomics"
562:
Phospholinked
Fluorescent Nucleotides or Real-time sequencing
111:
813:
1792:
Ronaghi, Mostafa; Uhlén, Mathias; Nyrén, Pål (1998-07-17).
1777:
DNA colony massively parallel sequencing ams98 presentation
1091:
The
Trustees of Columbia University in the City of New York
399:
that produces amplified copies of the single DNA template.
2013:"PacBio Announces Sequel Sequencing System - Bio-IT World"
1591:
1423:
Inc, Pacific Biosciences of California (October 3, 2013).
1365:
1248:
1939:
1768:
464:
407:
Amplification of a population of single DNA molecules by
57:
1528:
1476:
1308:
772:
1477:
Voelkerding KV, Dames S, Durtschi JD (September 2010).
364:
1794:"A Sequencing Method Based on Real-Time Pyrophosphate"
1464:"De novo bacterial genome assembly: a solved problem?"
624:
Nyren, P.; Pettersson, B.; Uhlen, M. (January 1993).
814:
Ballard D, Winkler-Galicki J, Wesoły J (July 2020).
773:
Voelkerding KV, Dames SA, Durtschi JD (April 2009).
623:
913:
402:
1653:
1920:"Fundamentals of 2 Base Encoding and Color Space"
1846:
1791:
862:
695:
544:Sequencing-by-ligation mediated by ligase enzymes
2032:
37:is any of several high-throughput approaches to
914:Tucker T, Marra M, Friedman JM (August 2009).
535:Sequencing by reversible terminator chemistry
64:. This design is very different from that of
1982:
965:"Next-generation sequencing: the race is on"
444:
415:DNA colony generation (Bridge amplification)
1678:
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701:Whitehead Institute for Biomedical Research
467:sample by detecting the incorporation of a
1544:
1542:
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1461:
458:
1704:
1674:
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1627:
1617:
1533:. Chichester: John Wiley & Sons, Ltd.
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980:
939:
890:
880:
839:
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745:
743:
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714:
653:
651:
295:Oligonucleotide 9-mer Unchained Ligation
1733:President and Fellows of Harvard College
1659:President and Fellows of Harvard College
1649:
1647:
453:
110:
96:but each utilizes a different strategy.
1548:
1537:
1244:
1242:
1166:
1164:
1106:
1104:
820:International Journal of Legal Medicine
691:
689:
255:Oligonucleotide 8-mer Chained Ligation
60:templates or single DNA molecules in a
14:
2033:
1669:
1524:
1522:
1304:
1302:
1081:
1079:
740:
711:
648:
570:can obtain sequence information while
338:Phospholinked Fluorescent Nucleotides
1787:
1785:
1762:
1679:Mitra RD, Church GM (December 1999).
1644:
863:Anderson MW, Schrijver I (May 2010).
1840:
1483:The Journal of Molecular Diagnostics
1239:
1161:
1101:
686:
619:
617:
615:
365:Template preparation methods for NGS
1531:Encyclopedia of Life Sciences (ELS)
1519:
1422:
1299:
1076:
24:
1782:
920:American Journal of Human Genetics
87:. Second, the DNA is sequenced by
25:
2052:
753:, Kawashima E, Farinellit L,
612:
503:
1897:. Helicosbio.com. Archived from
724:, Farinelli L, Kawashima E,
403:Gridded rolling circle nanoballs
75:
2005:
1991:
1976:
1933:
1912:
1887:
1862:
1743:
1721:
1585:
1470:
1455:
1416:
1359:
1213:
1051:
1034:"Specifications for HiSeq 2500"
1026:
1005:
956:
585:Clinical metagenomic sequencing
373:
275:Native dNTPs, proton detection
1983:Monica Heger (March 5, 2013).
907:
856:
807:
766:
520:real-time detection using the
309:Helicos Biosciences Heliscope
13:
1:
605:
516:activity (proof-reading) and
269:Life Technologies Ion Proton
229:Illumina Genome Analyzer IIX
119:HiSeq 2000 sequencing machine
35:massively parallel sequencing
1922:. Appliedbiosystems.cnpg.com
1810:10.1126/science.281.5375.363
963:von Bubnoff A (March 2008).
792:10.1373/clinchem.2008.112789
409:rolling circle amplification
232:Clonal Bridge Amplification
212:Clonal Bridge Amplification
192:Clonal Bridge Amplification
51:second-generation sequencing
7:
1462:Nederbragt L (2013-07-05).
595:Third-generation sequencing
590:First-generation sequencing
578:
31:Massive parallel sequencing
10:
2057:
1872:. Illumina. Archived from
1495:10.2353/jmoldx.2010.100043
982:10.1016/j.cell.2008.02.028
932:10.1016/j.ajhg.2009.06.022
832:10.1007/s00414-020-02294-0
315:Reversible Dye Terminator
235:Reversible Dye Terminator
215:Reversible Dye Terminator
195:Reversible Dye Terminator
43:next-generation sequencing
18:Next-generation sequencing
445:Single-molecule templates
428:on the surface. Repeated
249:Life Technologies SOLiD4
1551:Nature Reviews. Genetics
138:Max read length (bases)
1619:10.1073/pnas.1133470100
1429:GlobeNewswire News Room
1336:10.1126/science.1117389
1276:10.1126/science.1181498
660:Analytical Biochemistry
630:Analytical Biochemistry
459:Sequencing by synthesis
2041:DNA sequencing methods
1685:Nucleic Acids Research
672:10.1006/abio.1996.0432
642:10.1006/abio.1993.1024
292:Gridded DNA-nanoballs
120:
1751:US patent 8445194
1697:10.1093/nar/27.24.e34
1655:US patent 6485944
1189:10.1101/gr.091868.109
1087:US patent 7790869
454:Sequencing approaches
132:Template preparation
114:
882:10.3390/genes1010038
555:fluorescence imaging
477:amplification of DNA
2017:www.bio-itworld.com
1610:2003PNAS..100.8817D
1392:10.1038/nature11236
1384:2012Natur.487..190P
1327:2005Sci...309.1728S
1321:(5741): 1728–1732.
1267:2010Sci...327...78D
1227:on 30 December 2013
1137:10.1038/nature07517
1129:2008Natur.456...53B
567:Pacific Biosciences
330:Pacific Biosciences
125:
1954:10.1038/nmeth.2474
1870:"Assay Technology"
1447:has generic name (
779:Clinical Chemistry
514:3´to 5´exonuclease
289:Complete Genomics
123:
121:
1804:(5375): 363–365.
1604:(15): 8817–8822.
1378:(7406): 190–195.
508:The principle of
361:
355:
354:
345:); 30,000+ (max)
141:Run times (days)
102:genome sequencing
66:Sanger sequencing
16:(Redirected from
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2019:. Archived from
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1183:(9): 1527–1541.
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335:Single Molecule
312:Single Molecule
169:GS FLX Titanium
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2001:. October 2015.
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1307:
1300:
1261:(5961): 78–81.
1247:
1240:
1230:
1228:
1219:
1218:
1214:
1177:Genome Research
1169:
1162:
1123:(7218): 53–59.
1109:
1102:
1095:
1084:
1077:
1068:
1066:
1057:
1056:
1052:
1043:
1041:
1032:
1031:
1027:
1018:
1016:
1011:
1010:
1006:
961:
957:
912:
908:
861:
857:
812:
808:
771:
767:
760:
748:
741:
734:
719:
712:
705:
694:
687:
656:
649:
622:
613:
608:
581:
564:
546:
537:
506:
461:
456:
447:
417:
405:
392:oligonucleotide
376:
367:
358:
209:Illumina HiSeq
189:Illumina MiSeq
175:Pyrosequencing
155:Pyrosequencing
144:Max Gb per Run
78:
70:electrophoretic
28:
23:
22:
15:
12:
11:
5:
2054:
2044:
2043:
2027:
2026:
2023:on 2015-10-02.
2004:
1990:
1975:
1948:(6): 563–569.
1942:Nature Methods
1932:
1911:
1886:
1861:
1839:
1781:
1761:
1742:
1720:
1691:(24): 34e–34.
1668:
1643:
1584:
1536:
1518:
1489:(5): 539–551.
1469:
1454:
1415:
1358:
1298:
1238:
1212:
1160:
1100:
1075:
1050:
1025:
1004:
975:(5): 721–723.
955:
926:(2): 142–154.
906:
855:
806:
785:(4): 641–658.
765:
739:
710:
685:
647:
636:(1): 171–175.
610:
609:
607:
604:
603:
602:
597:
592:
587:
580:
577:
563:
560:
545:
542:
536:
533:
510:Pyrosequencing
505:
504:Pyrosequencing
502:
473:DNA polymerase
460:
457:
455:
452:
446:
443:
416:
413:
404:
401:
375:
372:
366:
363:
353:
352:
349:
346:
339:
336:
333:
326:
325:
322:
319:
316:
313:
310:
306:
305:
302:
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293:
290:
286:
285:
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279:
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273:
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246:
245:
242:
239:
236:
233:
230:
226:
225:
222:
219:
216:
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210:
206:
205:
202:
199:
196:
193:
190:
186:
185:
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179:
176:
173:
170:
166:
165:
162:
159:
156:
153:
150:
146:
145:
142:
139:
136:
133:
130:
124:NGS platforms
77:
74:
39:DNA sequencing
26:
9:
6:
4:
3:
2:
2053:
2042:
2039:
2038:
2036:
2022:
2018:
2014:
2008:
2000:
1994:
1986:
1979:
1971:
1967:
1963:
1959:
1955:
1951:
1947:
1943:
1936:
1921:
1915:
1901:on 2012-03-11
1900:
1896:
1890:
1876:on 2012-08-26
1875:
1871:
1865:
1853:
1849:
1843:
1835:
1831:
1827:
1823:
1819:
1815:
1811:
1807:
1803:
1799:
1795:
1788:
1786:
1778:
1774:
1773:
1765:
1752:
1746:
1734:
1730:
1724:
1716:
1712:
1707:
1702:
1698:
1694:
1690:
1686:
1682:
1675:
1673:
1660:
1656:
1650:
1648:
1639:
1635:
1630:
1625:
1620:
1615:
1611:
1607:
1603:
1599:
1595:
1588:
1580:
1576:
1572:
1568:
1564:
1560:
1556:
1552:
1545:
1543:
1541:
1532:
1525:
1523:
1514:
1510:
1505:
1500:
1496:
1492:
1488:
1484:
1480:
1473:
1465:
1458:
1450:
1438:
1430:
1426:
1419:
1411:
1407:
1402:
1397:
1393:
1389:
1385:
1381:
1377:
1373:
1369:
1362:
1354:
1350:
1346:
1342:
1337:
1332:
1328:
1324:
1320:
1316:
1312:
1305:
1303:
1294:
1290:
1286:
1282:
1277:
1272:
1268:
1264:
1260:
1256:
1252:
1245:
1243:
1226:
1222:
1221:"Ion Torrent"
1216:
1208:
1204:
1199:
1194:
1190:
1186:
1182:
1178:
1174:
1167:
1165:
1156:
1152:
1147:
1142:
1138:
1134:
1130:
1126:
1122:
1118:
1114:
1107:
1105:
1092:
1088:
1082:
1080:
1065:on 2014-11-06
1064:
1060:
1054:
1040:on 2014-12-06
1039:
1035:
1029:
1014:
1008:
1000:
996:
992:
988:
983:
978:
974:
970:
966:
959:
951:
947:
942:
937:
933:
929:
925:
921:
917:
910:
902:
898:
893:
888:
883:
878:
874:
870:
866:
859:
851:
847:
842:
837:
833:
829:
825:
821:
817:
810:
802:
798:
793:
788:
784:
780:
776:
769:
756:
752:
746:
744:
731:
727:
723:
717:
715:
702:
698:
692:
690:
681:
677:
673:
669:
665:
661:
654:
652:
643:
639:
635:
631:
627:
620:
618:
616:
611:
601:
598:
596:
593:
591:
588:
586:
583:
582:
576:
573:
572:phospholinked
568:
559:
556:
551:
541:
532:
530:
526:
523:
519:
515:
511:
501:
499:
495:
491:
487:
483:
478:
474:
470:
466:
451:
442:
440:
436:
431:
427:
423:
412:
410:
400:
398:
393:
388:
384:
381:
371:
362:
350:
347:
344:
340:
337:
334:
331:
328:
327:
323:
320:
317:
314:
311:
308:
307:
303:
300:
297:
294:
291:
288:
287:
283:
280:
277:
274:
272:Clonal-emPCR
271:
268:
267:
263:
260:
257:
254:
252:Clonal-emPCR
251:
248:
247:
243:
240:
237:
234:
231:
228:
227:
223:
220:
217:
214:
211:
208:
207:
203:
200:
197:
194:
191:
188:
187:
183:
180:
177:
174:
172:Clonal-emPCR
171:
168:
167:
163:
160:
157:
154:
152:Clonal-emPCR
151:
148:
147:
143:
140:
137:
134:
131:
128:
127:
118:
113:
109:
105:
103:
97:
94:
90:
86:
83:
76:NGS platforms
73:
71:
67:
63:
59:
54:
52:
48:
44:
40:
36:
32:
19:
2021:the original
2016:
2007:
1993:
1978:
1945:
1941:
1935:
1924:. Retrieved
1914:
1903:. Retrieved
1899:the original
1889:
1878:. Retrieved
1874:the original
1864:
1842:
1801:
1797:
1776:
1771:
1764:
1745:
1723:
1688:
1684:
1601:
1597:
1587:
1557:(1): 31–46.
1554:
1550:
1530:
1486:
1482:
1472:
1457:
1428:
1418:
1375:
1371:
1361:
1318:
1314:
1258:
1254:
1229:. Retrieved
1225:the original
1215:
1180:
1176:
1120:
1116:
1067:. Retrieved
1063:the original
1053:
1042:. Retrieved
1038:the original
1028:
1017:. Retrieved
1015:. Genome.gov
1007:
972:
968:
958:
923:
919:
909:
875:(1): 38–69.
872:
868:
858:
823:
819:
809:
782:
778:
768:
666:(1): 84–89.
663:
659:
633:
629:
600:RNA Velocity
565:
553:followed by
547:
538:
518:luminescence
507:
462:
448:
435:Pascal Mayer
430:denaturation
418:
406:
397:microreactor
377:
374:Emulsion PCR
368:
356:
106:
98:
79:
55:
50:
46:
42:
34:
30:
29:
1852:Solexa Ltd.
1445:|last=
730:Solexa Ltd.
439:Chris Adams
387:DNA library
385:methods, a
93:nucleotides
1926:2012-08-05
1905:2012-08-05
1880:2012-08-05
1848:US 6833246
1729:US 9624538
1069:2014-11-06
1044:2014-11-06
1019:2012-08-05
751:EP 0975802
722:EP 0972081
697:US 5641658
606:References
525:luciferase
490:IonTorrent
469:nucleotide
422:polymerase
164:0.40-0.60
149:Roche 454
135:Chemistry
1970:205421576
1818:0036-8075
1579:205484500
201:0.17-2.7
129:Platform
89:synthesis
62:flow cell
2035:Category
1962:23644548
1834:26331871
1715:10572186
1638:12857956
1571:19997069
1513:20805560
1437:cite web
1410:22785314
1353:11405973
1345:16081699
1293:17309571
1285:19892942
1207:19546169
1155:18987734
991:18329356
950:19679224
901:24710010
850:32451905
801:19246620
579:See also
494:Illumina
380:emulsion
341:10,000 (
117:Illumina
85:in vitro
1826:9705713
1798:Science
1606:Bibcode
1504:2928417
1401:3397394
1380:Bibcode
1323:Bibcode
1315:Science
1263:Bibcode
1255:Science
1198:2752135
1146:2581791
1125:Bibcode
999:8413828
941:2725244
892:3960862
841:7295846
755:Mayer P
726:Mayer P
680:8923969
529:apyrase
522:firefly
221:0.3-11
1968:
1960:
1857:
1832:
1824:
1816:
1757:
1738:
1713:
1706:148757
1703:
1664:
1636:
1629:166396
1626:
1577:
1569:
1511:
1501:
1408:
1398:
1372:Nature
1351:
1343:
1291:
1283:
1205:
1195:
1153:
1143:
1117:Nature
1096:
997:
989:
948:
938:
899:
889:
848:
838:
799:
761:
735:
706:
678:
550:ligase
486:PacBio
264:35-50
258:20-45
238:2x150
218:2x150
198:2x300
184:0.035
1966:S2CID
1830:S2CID
1575:S2CID
1349:S2CID
1289:S2CID
1231:1 Jan
995:S2CID
869:Genes
471:by a
426:oligo
348:0.08
332:SMRT
304:3000
298:7x10
241:2-14
224:1000
181:0.42
178:400‡
161:0.42
158:400‡
49:) or
1958:PMID
1822:PMID
1814:ISSN
1711:PMID
1634:PMID
1567:PMID
1509:PMID
1449:help
1406:PMID
1341:PMID
1281:PMID
1233:2014
1203:PMID
1151:PMID
987:PMID
969:Cell
946:PMID
897:PMID
846:PMID
797:PMID
676:PMID
496:and
351:0.5
318:35‡
284:100
281:0.5
278:200
261:4-7
1950:doi
1806:doi
1802:281
1701:PMC
1693:doi
1624:PMC
1614:doi
1602:100
1559:doi
1499:PMC
1491:doi
1396:PMC
1388:doi
1376:487
1331:doi
1319:309
1271:doi
1259:327
1193:PMC
1185:doi
1141:PMC
1133:doi
1121:456
977:doi
973:132
936:PMC
928:doi
887:PMC
877:doi
836:PMC
828:doi
824:134
787:doi
668:doi
664:242
638:doi
634:208
498:MGI
482:454
465:DNA
383:PCR
378:In
343:N50
324:25
301:11
244:95
204:15
115:An
82:PCR
58:DNA
47:NGS
33:or
2037::
2015:.
1964:.
1956:.
1946:10
1944:.
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1800:.
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