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

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…

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 __could add picture of fragment and read

Full Genome Re-sequencing has been done for many cancers and rare clinical disorders Many times mutations have been found in regulatory regions

Exome sequencing is a cost-effective to identify de novo protein coding mutations __ show how exome libraries are prepared exome libraries are prepared by capture on an array of synthetic oligos

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

RNA-seq

ChIP-seq

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

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

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

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

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

Roche 454 Sequencing Metzker, NG 2010

Roche 454 Sequencing

Roche 454 Peak Heights Data

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

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

Illumina (Solexa) Genome Analyzer and Flow Cell

Illumina On-Chip Amplification

Illumina (Solexa) Sequencing

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

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

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

SOLiD Sequencing by Oligonucleotide Ligation and Detection

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

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

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.

ABI SOLiD Sequencing Cycle

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

SOLiD Color Coding Scheme Blue is color of homopolymer runs Mapping into color space 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

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

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

An Ion Torrent Chip

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

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.

Ion Torrent Advantages & Drawbacks Loading Density 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

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

Newer Technologies Complete Genomics Pacific Biosciences Oxford Nanopore

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

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

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