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High-Throughput Sequencing Technologies Biological Sequence Analysis BNFO 691/602 Spring 2014 Mark Reimers.

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Presentation on theme: "High-Throughput Sequencing Technologies Biological Sequence Analysis BNFO 691/602 Spring 2014 Mark Reimers."— Presentation transcript:

1 High-Throughput Sequencing Technologies Biological Sequence Analysis BNFO 691/602 Spring 2014 Mark Reimers

2 Outline What can we do with next-generation sequencing? –De novo sequencing of simple genomes –Re-sequence individual variations –Generate genome-wide quantitative data for a variety of assays What technologies are now available and which are up-and-coming? –Roche, Illumina, SOLiD, Ion Torrent, etc…

3 What is High-Throughput Sequencing? Generating many thousands or millions of short (30 to 1,000 base) sequences by sequencing parts of longer (200+ base) DNA fragments Most research uses reads from one end of a fragment (single-end), but most technologies can be adapted to make paired-end reads on opposite strands

4 Full Genome Re-sequencing has been done for many cancers and rare clinical disorders

5 Exome sequencing is a cost-effective to identify de novo protein coding mutations

6 Targeted re-sequencing of a few relevant genes can identify diverse critical mutations across a large number of cases

7 RNA-seq

8 ChIP-seq

9 DNA methylation profiling mC  C C  U C  C U  T After PCR PCR+Seq

10 DNAse Hyper-sensitivity DNAse I enzyme cuts DNA Much more likely to cut at open chromatin Two approaches: –Cut slowly then fragment and sequence ends –Cut rapidly then sequence short fragments

11 Mapping of chromatin interactions (5C) (courtesy Elemento lab)

12 HTS Technologies Roche-454 Illumina SOLiD Ion Torrent Newer Technologies Outlook

13 Founded by Jonathan Rothberg as a secret project (code-named ‘454’) within CuraGen

14 Roche 454 Sequencing Metzker, NG 2010

15 Roche 454 Sequencing

16 Roche 454 Peak Heights Data

17 Advantages & Drawbacks PRO Long reads are uniquely identifiable Relatively quick ~20 hours total CON Cost is relatively high Frequent errors in runs of bases Frequent G-A transitions

18 Best Uses of Roche 454 De novo small genome (prokaryote or small eukaryote genome) sequencing Metagenomics by16S profiling Used to be best for metagenomics by random sequencing –new long reads from Illumina are competitive Targeted re-sequencing of small samples

19

20 Illumina (Solexa) Genome Analyzer and Flow Cell

21 Illumina On-Chip Amplification

22 Illumina (Solexa) Sequencing

23 Paired-End Illumina Method Paired-end reads are easy on Illumina because the clusters are generated by ligated linkers. Different linkers and primers are attached to each end

24 Advantages & Drawbacks PRO –Very high throughput –Most widespread technology so that comparisons seem easier CON –Sequencing representation biases, especially at beginning –Slow – up to a week for a run

25 Best Uses of Illumina Expression analysis (RNA-Seq) Chromatin Immunoprecipitation (ChIP- Seq) Metagenomics by random sequencing

26 SOLiD Sequencing by Oligonucleotide Ligation and Detection

27 SOLiD History George Church licensed his ‘polony’ technique to Agencourt Personal Genomics ABI acquired the SOLiD technology from Agencourt in 2006

28 SOLiD Preparation Steps Prepare either single or ‘mate-pair’ library from DNA fragments Attach library molecules to beads; amplify library by emulsion PCR Modify 3’ ends of clones; attach beads to surface

29 Emulsion PCR Emulsion PCR isolates individual DNA molecules along with primer-coated beads in aqueous droplets within an oil phase. A polymerase chain reaction (PCR) then coats each bead with clonal copies of the DNA molecule. The bead is immobilized for sequencing.

30

31 ABI SOLiD Sequencing Cycle

32 SOLiD Reads Each Base Twice Most bases are matched by two primers in different ligation cycles

33 SOLiD Color Coding Scheme Blue is color of homopolymer runs If you translate color reads directly into base reads then every sequence with an error in the color calls will result in a frame-shift of the base calls. it is best to convert the reference sequence into color-space. There is one unambiguous conversion of a base reference sequence into color-space, but there are four possible conversions of a color string into base strings

34 Advantages & Drawbacks PRO –Very high throughput –Di-base ligation ensures built-in accuracy check Low error rate for low-coverage –Can handle repetitive regions easily CON –Strong cycle-dependent biases (can be modeled and partly overcome – see Wu et al, Nature Methods, 2011) –Low quality color calls (Phred < 20) are common –Reported problems with paired ends – most mapped tags don’t map to the same chromosome

35

36 Ion Torrent Sample Prep Emulsion PCR loads copies of unique sequences onto beads One bead is deposited in each well of a micro-machined plate

37 An Ion Torrent Chip

38 When a nucleotide is incorporated into a strand of DNA by a polymerase, a hydrogen ion is released From promotional material

39 Ion Torrent Sequencing Process As in 454, nucleotides are washed over the nascent strand in a prescribed sequence. Each time a nucleotide is incorporated, hydrogen ions are released and detected.

40 Ion Torrent Advantages & Drawbacks PRO –Very high throughput potential –Very fast (an afternoon) CON –Homopolymer runs are still a problem –Very uneven loading of sequences wastes a lot of real estate on the chips –No prospect of paired-ends –Not many applications yet for their error model Loading Density

41 Newest Machine – Ion Proton $150K per machine Ion Proton I chip has 165 million sensors –Intended for exomes Ion Proton II chip has 660 million sensors –50X more than 318 chip –Claim $1K genome this year

42 Newer Technologies Complete Genomics Pacific Biosciences Oxford Nanopore

43 Complete Genomics Service company only – no equipment sales ~$4,000 per human genome (2011 price) DNA Nanoball technology generates paired-end sequences plated at high density Sequenced by ligation

44 Pacific Biosciences Single-molecule real-time (SMRT) sequencing by circular strand technology using semiconductor technology Long reads promised at under $200 per genome High error rates reported

45 Oxford Nanopore Single-molecule sequencing by threading DNA through a protein nanopore GridION is a general technology for sequencing polymers by measuring current – can do polypeptides also


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