Next Generation Sequencing

Exome Sequencing

Marcela Davila Genomics Core Facility

NGS methods First generation (great cost, intense human effort) 1954 – Sequencing by degradation (Whitfeld PR) 1975 – Chain termination method (Sanger & Coulson) 1977 – Chemical modification (Maxam and Gilbert) Second generation (sincronyzed washing/scanning) SBS – Illumina Pyrosequencing – Roche SBL – AB SOLiD Third generation (increase sequencing speed, high throughput, no optics) Semiconductor: Ion Torrent SBS-single molecule: Helicos SBS-single molecule-real time: Pacific Biosciences SBH/SBL- Complete Genomics FRET: VisiGen Protein nanopores: Oxford Nanopore TEM: Halcyon Molecular and ZS Genetics Transistor mediated: IBM STM: Reveo

Sanger method Dye-labeled terminator

Capillar electrophoresis

DNA template

Laser beam Chromatogram

Next generation sequencing For cyclic array sequencing 1. 2. 3. 4. 5.

DNA library preparation (ligation of adapters) Amplification (ePCR, bridge PCR) Sequencing reaction Imaging Decoding

Next generation sequencing For cyclic array sequencing 1. 2. 3. 4. 5.

DNA library preparation (ligation of adapters) Amplification (ePCR, bridge PCR) Sequencing reaction Imaging Decoding

Next generation sequencing For cyclic array sequencing 1. 2. 3. 4. 5.

DNA library preparation (ligation of adapters) Amplification (ePCR, bridge PCR) Sequencing reaction Imaging Decoding

SeqBySynthesis - Illumina

Pyrosequencing - Roche

Pyrogram

SeqByLigation – AB/SOLiD

First round

SeqByLigation – AB/SOLiD Second round

SeqByLigation – AB/SOLiD

IonSensitiveFieldEffectTransistors – Ion Torrent

SeqBySynthesis - single molecule - Helicos 1 2 3 A

A

C

C

G

G G G

T

T

A

C

A

C G T

C G T

Single Molecule Real Time – Pacific Biosciences

combinatorialProbeAnchorLigation – Complete Genomics

FluorescenceResonanceEnergyTransfer – VisiGen

Protein nanopores – Oxford Nanopore Tech

Exonuclease

Transmission Electron Microscopy – Halcyon Molecular/ZS Genetics

Electronic fingerprint

STM - Transistor mediated – IBM

metal

Dielectric layers

ScanningTunnelingMicroscope– Reveo

References Niedringhaus TP, et al (2011) Metzker ML (2010) Schadt EE, et al (2010) Tanaka H and Kawai T (2009) Drmanac R, et al (2009) Mardis ER. (2008) http://www.illumina.com/Media/flash_player.ilmn?dirname=systems&swfname=GA_ workflow_vid&width=780&height=485&iframe http://my454.com/products/technology.asp http://appliedbiosystems.cnpg.com/Video/flatFiles/699/index.aspx http://www.helicosbio.com/Technology/TrueSingleMoleculeSequencing/tSMStradeH owItWorks/tabid/162/Default.aspx http://www.pacificbiosciences.com/aboutus/videogallery?videoImage=pac_bio_lg.jpg http://www.nanoporetech.com/news/movies#movie-24-nanopore-dna-sequencing http://www.invitrogen.com/site/us/en/home/Products-andServices/Applications/Sequencing/Semiconductor-Sequencing/SemiconductorSequencing-Technology/Ion-Torrent-Technology-How-Does-It-Work.html http://www.abrf.org/Other/ABRFMeetings/ABRF2005/Hardin.pdf http://researcher.ibm.com/view_project.php?id=1120

Different applications, different pipelines Quality Check Quality Filter Mapping to reference genome Realignment and recalibration

Resequencing

SNV detection

RNA-seq

Transcript abundance estimation

ChIP-Seq

Peak detection

SNPs Human genome 3 billion bps

AAG-TA AAGCTA

UTRs

3 million differences (0.1%)

AAGCCTA AAGCTTA

Coding regions UAG GGU ACU * G T

AAGCTA AAG-TA

Splice sites/branch site

Targeted resequencing DNA library

Hybridization

Capture

Biotin probes

Streptavidin beads

Different recipies R1

Single end (SE) R1

R2

Paired-end (PE) 200-500 bp

R1

R2

Mate-pair (MP) 2-5 Kb

Fastq format R2

R1

@HWI-H200:53:D08U2ACXX:5:1101:1231:2012 1:N:0: GCATTTTAGTAGAACCAGNCATTTCCCCCNACNTCNNTNCGNNANNNNTAA + @CCFFFFFHFFHHJJJJJ#3 TGA = W->X change have? TGA -> CGA = X->R Damaging, benign

the AA

Is it a known SNP?

Variants list

300,000 SNPs - 10,000 Indels

Variants filtering Exome sequencing cases Family filters

Coding variants

http://www.sciencedirect.com/science/article/pii/S0002929711003946

Disease model Disease knowledge

Controls Genetic variation DBs

The real work begins…

Candidate genes

Data visualization