DNA Sequencing by Ion Torrent. Marc Lavergne CHEM 4590

Transcription

1 DNA Sequencing by Ion Torrent Marc Lavergne CHEM 4590

2 OVERVIEW History DNA Synthesis and First-Gen Sequencing Technology Sequencing Signal Detection Advantages/Disadvantages Applications Current Research Studies

3 DEVELOPMENT HISTORY First-gen sequencing Sanger Sequencing Required: DNA template DNA primer DNApol Modified ddntp Radioactive tags Fluorescent tags Sequence ladder by radioactive sequencing compared to fluorescent peaks

4 HISTORY: DNA SYNTHESIS In nature the incorporation of a dntp into a growing DNA strand involves the formation of a covalent bond and the release of pyrophosphate and a hydrogen ion

5 HISTORY: DNA SYNTHESIS A dntp is only incorporated if it is complementary to the leading unpaired template nucleotide Ion torrent exploits this by determining if a hydrogen ion is released after adding a single species of dntp to the reaction

6 TECHNOLOGY: SEQUENCING How does Ion Torrent work? Microwells on a semiconductor chip contain many copies of 1 single-stranded template DNA that we want to sequence The sequence is flooded by DNA polymerase and unmodified dntp (A, C, G, or T) IF a dntp is complementary to the template strand it is added to growing complementary strand by the DNApol IF NOT no incorporation = no biochemical reaction

7 TECHNOLOGY: SIGNAL DETECTION

8 TECHNOLOGY: SIGNAL DETECTION Utilizes an ISFET Ion-sensitive field-effect transistor Measures ion concentration in solution

9 ION TORRENT STRENGTHS Rapid sequencing speed Low price and operating cost Sequencing occurs in real-time Event timeline Incorporation measurement = 4 seconds Individual run = 1 hour Times can be reduced as chip technology continues to develop

10 ION TORRENT WEAKNESSES dntp homopolymer repeats Difficult to enumerate long repeats E.g. Homorepeats of length 7 can t be differentiated from length 8 A common limitation shared by other techniques that detect single nucleotide additions E.g. pyrosequencing Short read length Avg. ~400 nucleotides per read Less than pyrosequencing and Sanger sequencing Longer read lengths are beneficial for de novo genome assembly

11 COMPARISON OF HIGH-THROUGHPUT SEQUENCING METHODS Method Ion Torrent Pyro sequencing (454) Illumina (Sequencing by synthesis) Nanopore Sanger Sequencing (Chain Termination) Read Length Up to 600bp Accuracy Reads per Run Time Per Run Cost per 1million bases ($USD) Advantages 98% >80 million 2 hours $1 Fast. Low equipment cost 700bp 99.9% 1 million 24 hours $10 Long read size. Fast * bp Up to 500 kb bp 99.9% *1 million to 3 billion ~92-97% ind. Single read 99.96% concensus Variable 1-11 Days $0.05 to $015 Real time Data Collection (1min 48hr) 99.9% N/A 20min to 3 hr $500- $1000 per cell High sequence yield Longest indv reads. Portable technology. $2400 Wide application Disadvantages Homopolymer errors Expensive runs. Homopolymer errors Very expensive equipment. Requires High DNA conc. Lower output. Low single read accuracy Expensive and impractical for larger sequences. Requires time to clone plasmid or PCR

12 APPLICATION Rapid, compact sequencer Easily utilized as bench top machine Developers want to take sequencing outside of specialized centers Small read length = best suited to small scale application Developers want to change this by increasing the density of the chips Ion Torrent Personal Genome Machine - Thermo Fisher Scientific. Business Wire, 2 June 2014, Organizations-Adopt-Ion-Torrent-Next-Generation.

13 Current Studies

14 NGS tools for traceability in candies as high processed food products First time PGM (ion torrent) is used to evaluate food traceabiliy of high processed products Explored the applicability of NGS methods to analyze the composition of different types of candies as examples of highly processed food products. Ion Torrent Personal Genome Machine (PGM) used to amplify a short fragment of mitochondrial 16S ribosomal gene, and compared the results with previous data obtained from classic PCR-cloning-sequencing methodology.

15 NGS tools for traceability in candies as high processed food products Highly processed products are one of the challenges of food traceability Proteins and DNA are generally degraded due to such intense processing. Short DNA amplicons are sought for successful analysis As well as DNA fragments that are naturally in multiple copies in living organisms, For example mitochondrial genes

16 NGS tools for traceability in candies as high processed food products Fig. 2. Proportion of sequences detected for each species in the total data from PGM (A), and proportion of candies where each species appeared in the analyses (B).

17 NGS tools for traceability in candies as high processed food products Table 1. Classification of the analyzed candy products based on the number of species detected in them. PCR-cloning-sequencing as PCR-CS, Ion Torrent Personal Genome Machine as PGM. Bulk PCR-CS PGM Packed PCR-CS PGM None species species species species >4 species

18 Any questions?