Presentation on theme: "Current Sequencing Technologies and Data Generation Corbin Jones & Piotr Mieczkowski Department of Biology, College of Arts and Sciences, Carolina Center."— Presentation transcript:
Current Sequencing Technologies and Data Generation Corbin Jones & Piotr Mieczkowski Department of Biology, College of Arts and Sciences, Carolina Center for Genome Sciences Department of Genetics, School of Medicine, University of North Carolina at Chapel Hill
Library prep Sequencing
Sample Submission Sonication QC LIMS End Repair Adenylation Adapter Ligation Size Selection PCR QC 15 nM dilution Pooling for multiplexing Facility Sample flow Transfer to new plate/tube Data flow 3 µl of Sample Sample failed y/n Concentration Size Leftover sample Dilution Leftover sample SAMPLE Sample flow Same plate
Manual and Semiautomation in HTSF library prep workflow Sonication Sage – Pippin – Automated size selection system Automated library size selection Magnetic beads DNA size selection
Automation in HTSF Tecan – Freedom Evo system – 8 tip -2x48 (96) samples per week – DNA library prep -Automated sample normalization steps -PCR and qPCR preparation -Reagans distribution -Can be adapted to small and medium scale protocols for Illumina and Ion Torrent -We have all necessary components for DNA/RNA extraction using Qiagen kits Caliper – Sciclone system 96 tip pipetting head (8-96 samples per run) -TruSeq DNA library preparation -TruSeq Exome Enrichment -SureSelect Agilent DNA capture and library preparation
N EXT - GENERATION S EQUENCING (D EEP S EQUENCING ) P LATFORMS o Short reads 1. Genome Analyzer IIx (GAIIx), HiSeq2000, HiSeq2500, MiSeq – Illumina 2. SOLiD 5500xl System – Applied Biosystem 3. HeliScope™ Single Molecule Sequencer - Helicos o Long reads 1. Genome Sequencer FLX System (454) – Roche 2. PacBio RS - Pacific Bioscience 3. Personal Genome Machine, Ion Proton - Ion Torrent 4. GridION – Oxford Nanopore o Mapping sequences to large DNA fragments 1. NABsys 2. Bionanomatrix
UNC – HTSF 9 HiSeq 2000/ GA II PacBio Ion Torrent MiSeq (Jeff Dangl) Liz Buda and Donghui Tan Also on campus: 454 (Microbiome) 454 jr. (Viral genomics) MiSeq – Kevin Weeks
What type of sequencing should I choose for the Illumina sequencing project? HiSeq 2000/2500 – mln single end sequencing reads per lane. - ChIPseq – Single End 50 cycles (2-3 human samples per lane) - RNAseq – Single End 50 cycles (2-3 human samples per lane) If you are interested in splicing variants and fusion genes both Single End 100cycles and Paired End 2x50cycles will be better option for you. -Whole Genome Sequencing – Paired End 2x100cycles (2-3 lanes per genome) -Exome Capture - Paired End 2x100cycles (4 samples per lane) MiSeq – 3-7 mln single end sequencing reads per lane. Custom projects, fast turnaround. Metagenomics - 16S profile – Paired End 2x150cycles up to 24 samples per lane. -Whole Microbial Genome Sequencing - Paired End 2x150cycles
HiSeq 2000 SHORT READ PLATFORMS at UNC Initially capable of up to 600Gb per run in 13 days. Cost of resequencing one human genome: Now UNC PI - (30x coverage) about $6,000 Now for outside of UNC - (30x coverage) about $9,000 HiSeq 2500 Initially capable of up 100Gb per run in 27hours. Cost per genome - ???
MiSeq -Small capacity system. PE 2x150cycles in 27hours. -PE 2 x 250bp coming soon – error rate for read 1 – less than 1%; read 2 about 1.2%. -In preparation – PE 2 x 400bp – error rate for read1 about 2%; read 2 about 4%. -In preparation – Longer insert size possible 1.5kb
PacBio RS Single molecule resolution in real time Short waiting time for result and simple workflow – Generate basecalls in <1 day – Polymerase speed ≥1 base per second No amplification required – Bias not introduced – More uniform coverage Direct observation – Distinguish heterogeneous samples – Simultaneous kinetic measurements Long reads – Identify repeats and structural variants – Less coverage required Information content – One assay, multiple applications Genetic variation (SVs to SNPs) Methylation Enzymology C2 chemistry – installed March Long reads 6-10kb -Meidan size of molecules 3kb -Still 15% error rate -No strobe sequencing Software focus on: De novo assembly Hi quality CCS consensus reads In preparation -Load long molecules by magnetic beads -Modified nucleotides detection
Sample Preparation LS – long sequencing reads Large insert sizes (2kb-10kb) Generates one pass on each molecule sequenced Small insert sizes 500bp Generates multiple passes on each molecule sequenced Standard Circular Consensus CCS – high quality sequencing reads PacBio RS – two sequencing modes
Example Data: 1 smart cell Pre-Filter # of Bases 180,320,136 bp Post-Filter # of Bases 165,424,592 bp Pre-Filter # of Reads 75153Post-Filter # of Reads Pre-Filter Mean Readlength 2399 bp Post-Filter Mean Readlength 3133 bp Pre-Filter Mean Read Quality Post-Filter Mean Read Quality % Adapter Dimer (0-10bp) 1.94 % % Short Insert (11-100bp) 0.47 %
Personal Genome Machine – Ion Torrent (life technologies) Three types of semiconductor chips: 314 – 20Mb Mb 318 – 1Gb Read length depends on base composition bp (200cycles) System is enabled for Paired End 2x100cycles The fastest sequencing system on the market. Recommendation: Resequencing applications which require fast turnaround of samples - Amplicons (PCR products) -Small and medium size genomes -Custom DNA capture applications How it works : H+ ion is released during base incorporation. Individual polymerases attached to beads are positioned in tiny wells that rest on a tiny pH meter. How it works : H+ ion is released during base incorporation. Individual polymerases attached to beads are positioned in tiny wells that rest on a tiny pH meter.
PGM/Ion Torrent Data 316 chip Thr. Total Number of Bases [Mbp] ‣ Number of Q17 Bases [Mbp] ‣ Number of Q20 Bases [Mbp] Total Number of Reads 368,860 Mean Length [bp] 211 Longest Read [bp] 380
Library Preparation from Low Quantities of DNA or RNA Mondrian SP System – NuGEN Technologies -Human libraries from 5ng of total DNA. Only 10-15% of duplicate reads. -Ultralow DNA library systems Soon: -Ultralow RNA library systems -Libraries from total RNA with rRNA depletion. Advanced Liquid Logic from RTP Microfluidics stationary and portable systems
Ion Proton System -Human genome in one day -Cost of reagents $1000 per run -Error rate around 1.2% -Human Genome, RNAseq, ChIPseq Ion Proton Chip I – 10Gb (Whole Exome capture experiments) Ion Proton Chip II – 100Gb Whole human Genome resequencing
Oxford Nanopore – new view on sequencing Hemolysin – pore - inner diameter of 1nm, about 100,000 times smaller than that of a human hair.
Oxford Nanopore DNA sequencing Error rate 4%, prediction for end of the year 0.1 – 2%.
Oxford Nanopore – new concepts MinION - 150Mb per run - Tested 48kb read length -$900 per instrument -500 pores per device GridION - XXXMb per run - Tested 48kb read length -$XXX per instrument pores per device, soon 8000 pores -Cost per human genome $1500.
Oxford Nanopore – applications -DNA sequencing -Protein detection -Protein DNA interaction -Small molecule detection -96 well plates for 96 samples -Controlled time of sequencing
Intelligent BioSystems Mini20 System (manufactured by Azco Biotech) Amplification by rolony method Sequencing by Synthesis with announced 100 base reads, but expect to compete with Sanger down the road Designed for clinical labs 20 independent flow cells, no queue for loading, run asynchronously 20M reads/flow cell, 4 GB/ flow cell Potential problems with repeats System cost $120K, $150 flow cell (disposable), full costs per sample not clear yet. Entering early access now, expect commercial shipping late 2012
Genia Technologies Very early stage announcement – Backed by Life Technologies (at least 1 year away) Describe system as a cross between Ion Torrent and Oxford Nanopore Electronic “Active Control” technology enables highly efficient nanopore-membrane assembly and control of DNA movement through the channel Initially used α-Hemolysin and claimed 98% raw accuracy with that but now are using an undisclosed pore for further development. Claim sensitivity 1-2 orders of magnitude greater than Oxford Nanopore. Ramping up pore density to 100K pores/chip by end of Plan to market a mobile reader for <$1K and per sample costs <$100 Plan early access in late 2012, commercial shipment 2013