Accurate multiplex polony sequencing of an evolved bacterial genome j shendure, gj porreca, nb reppas, x lin, jp mccutcheon. Oct 21, 2011 accurate multiplex polony sequencing of an evolved bacterial genome. Millions of beads were immobilized in a polyacrylamide gel and subjected to automated cycles of sequencing by ligation and fourcolor imaging. Sponsored by this booklet is brought to you by the aaas science business office. A sheared, sizeselected genomic fragments yellow are circularized with a linker red bearing mme i recognition sites note s1. Initial idea of polony polymerase colony sequencing was developed by george church accurate multiplex polony sequencing of an evolved bacterial genome jay shendure, gregory j.
To minimize efforts, it can be desirable to pool samples prior to target capture to perform both capture and sequencing in a multiplex setup. Science 3095741, 1728 1732 sequence and structural variation science 3095741, 1728 1732 sequence. Genome accurate multiplex polony sequencing of an evolved. Polony sequencing technology was initially developed with the aim to address the cost efficiency of genome resequencing. To demonstrate accurate sequencing of human dna, we sequenced a human bacterial artificial chromosome bac clone bcx98j21 that contained 162,752 bp of the major histocompatibility. A new microarraybased method of wholegenome resequencing called comparative genome sequencing. Genomics bacterial genome sequencing and annotation. Multiplexing strategies for microbial whole genome smrt sequencing author. We describe a dna sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic dna sequencing automation. Each offers a different compromise between the length of reads, which determines what proportion of the genome data can be uniquely mapped to, and depth of coverage, which determines the dynamic range over which gene expression can be quantified.
It soon followed by the alternative approaches of massively parallel sequencing based on multiplex polony sequencing, pyrosequencing 454 roche, inc, reversible dyetermination genome analyzer by. A cellfree, matepaired library provided single dna molecules that were amplified in parallel to 1micrometer beads by emulsion polymerase chain reaction. This automated nonelectrophoretic dna sequencing method comprises four basic steps that include construction of an in vitro pairedtag library from genomic dna, generation of polonies by clonal amplification of library molecules on microbeads by emulsion polymerase chain. The world has now entered into a new era of genomics because of the continued advancements in the next generation high throughput sequencing technologies, which includes sequencing by synthesisfluorescent in situ sequencing fisseq, pyrosequencing, sequencing by ligation using polony amplification, supported oligonucleotide detection solid, sequencing by hybridization along with. The human genome project hgp was an international scientific effort to sequence the entire human genome, that is, to produce a map of the base pairs of dna in the human chromosomes, most of. Multiplex amplification of large sets of human exons. Polony sequencing is an inexpensive but highly accurate multiplex sequencing technique that can be used to read millions of immobilized dna sequences in parallel. Accurate multiplex polony sequencing of an evolved bacterial genome shendure, jay. Binnewies 1, heng wu 1, thomas sicheritzponten 1, hanni willenbrock 1, david w. Church helped to develop and refine techniques with which to describe the. Polony multiplex analysis of gene expression pmage in mouse hypertrophic cardiomyopathy.
Double indexing overcomes inaccuracies in multiplex. From genetics to genomics of epilepsy pubmed central pmc. Massive parallel sequencing or massively parallel sequencing is any of several highthroughput approaches to dna sequencing using the concept of massively parallel processing. Sep 22, 2017 shendure j, porreca gj, reppas nb, lin x, mccutcheon jp et al 2005 accurate multiplex polony sequencing of an evolved bacterial genome. George mcdonald church studied dna from living and from extinct species in the us during the twentieth and twentyfirst centuries. Abstract we describe a dna sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic dna sequencing automation. We describe a dna sequencing technology in which a commonly. Genome sequencing in microfabricated highdensity picolitre reactors. Cost per base was roughly oneninth as much as that of. Sequencing, assembly, and annotation of the soybean genome. Subsequent steps, which include a rolling circle amplification, yield the 4 to 6bp matepaired library molecules shown at right. Shendure j1, porreca gj, reppas nb, lin x, mccutcheon jp, rosenbaum am, wang md, zhang k, mitra rd, church.
Using 36 base fragment and 26 base jumping reads from five microbial. In many cases, sequencing libraries need to be enriched for certain targets of interest, often by means of hybridization capture, before they are subjected to multiplex sequencing. While illumina data is appropriate for many analyses, its short read length limits its ability to resolve genomic. Genome technology program bibliography publications and patents nhgri launched an ambitious program in 2004 to reduce the cost of full genome sequencing initially by 100fold and subsequently by an additional 100fold. Request pdf on researchgate accurate multiplex polony sequencing of an evolved bacterial genome we describe a dna sequencing technology in which. Shendure j1, porreca gj, reppas nb, lin x, mccutcheon jp, rosenbaum am, wang md, zhang k, mitra rd, church gm. We apply this technology to resequence an evolved strain. Shendure j, porreca gj, reppas nb, lin x, mccutcheon jp, rosenbaum am, wang md, zhang k, mitra rd, church gm 2005 accurate multiplex polony sequencing of an evolved bacterial genome. Ems mutagenesis and point mutation discovery request pdf. Shendure j, porreca gj, reppas nb, lin x, mccutcheon jp et al 2005 accurate multiplex polony sequencing of an evolved bacterial genome. Multiplexing strategies for microbial whole genome smrt. Accurate whole human genome sequencing using reversible. Accurate multiplex polony sequencing of an evolved bacterial genome jay shendure,1.
To demonstrate accurate sequencing of human dna, we sequenced a human bacterial artificial chromosome bac clone bcx98j21 that contained 162,752 bp of the major histocompatibility complex on human chromosome 6 accession al662825. Accurate multiplex polony sequencing of an evolved bacterial. To demonstrate accurate sequencing of human dna, we sequenced a human bacterial artificial chromosome bac clone bcx98j21 that contained 162,752 bp of the major histocompatibility complex on. Sep 09, 2005 accurate multiplex polony sequencing of an evolved bacterial genome. Illumina sequencing platforms have enabled widespread bacterial whole genome sequencing. Modern dna sequencing began in 1977, with development of the chemical method of maxam and gilbert and the dideoxy method of sanger, nicklen and coulson, and with the first complete dna sequence phage x174, which demonstrated that sequence could give profound insights into genetic. Comparative genome sequencing of escherichia coli allows.
This automated nonelectrophoretic dna sequencing method comprises four. The pcr amplifications before sequencing are potentially introducing errors at a rate that imposes a ceiling on the accuracies. As the throughput of the pacbio systems continues to increase, so has the desire to fully\r utilize smrt cell sequencing capacity to multiplex microbes for whole genome sequencing. Personal genome sequencing with complete genomics technology. While illumina data is appropriate for many analyses, its short read length limits its ability. Glick department of biology, university of waterloo, 200 university avenue west, waterloo, ontario n2l 3g1, canada since the first. Science 3095741, 1728 1732 sequence and structural variation science 3095741, 1728 1732 sequence and structural variation. Illumina, 454 and solid sequencing platforms have been used in bacterial rnaseq studies 27, 28, 29. The embryo project encyclopedia sequence analysis, dna. 2005 by j shendure, g j porreca, n b reppas, x lin, j p mccutcheon, a m rosenbaum, zhang wang, k mitra, r d. Highly multiplexed subcellular rna sequencing in situ. Genome technology program bibliography publications and patents nhgri launched an ambitious program in 2004 to reduce the cost of full genome sequencing initially by 100fold and subsequently.
The threedimensional architecture of a bacterial genome. Below, listed by principal investigator, are citations and links to many of the. Noninvasive wholegenome sequencing of a human fetus. Double indexing overcomes inaccuracies in multiplex sequencing on the illumina platform. Each offers a different compromise between the length of reads, which determines what proportion of. Modern dna sequencing began in 1977, with development of the chemical method of maxam. Shendure j, porreca gj, reppas nb, lin x, mccutcheon jp, rosenbaum am, wang md, zhang k, mitra rd, church gm 2005. We demonstrate that genome sequences approaching finished quality can be generated from short paired reads. George mcdonald church 1954 the embryo project encyclopedia.
Abstract we describe a dna sequencing technology in which a commonly available, inexpensive. George churchs laboratory at harvard university has developed a high throughput technology based on polony amplification and fisseq. Completing bacterial genome assemblies with multiplex. Accurate multiplex polony sequencing of an evolved bacterial genome a reverse genetic, nontransgenic approach to wheat crop improvement by tilling jan 2005 1728173275. Porreca and nikos basil reppas and xiaoxia lin and john p. The threedimensional architecture of a bacterial genome and. Accurate multiplex polony sequencing of an evolved bacterial genome jay shendure, gregory j. Accurate multiplex polony sequencing of an evolved bacterial genome article in science 3095741. Studying bacterial transcriptomes using rnaseq sciencedirect. Jun 12, 2019 accurate multiplex polony sequencing of an evolved bacterial genome. Church1 we describe a dna sequencing technology in which a commonly available.