SNPs and Human Diseases XV Robert Kraaij Department of Internal - - PowerPoint PPT Presentation

DepthOfCoverage

NGS technologies SNPs and Human Diseases XV

Robert Kraaij Department of Internal Medicine r.kraaij@erasmusmc.nl

What will NGS bring us?

RFLP TaqMan Array Array and Imputation Regional Sequencing Full Genome Sequencing

• First Generation: a bit of history
• Next (Second) Generation
• Third Generation

1977: Maxam & Gilbert Sequencing

Walter Gilbert from wikipedia.org

Maxam & Gilbert Sequencing

G G+A C+T C

1977: Sanger Sequencing

Frederick Sanger from wikipedia.org

G A T C

Sanger Sequencing

Sanger sequencing landmarks

from wikipedia.org

• 1977

bacteriophage φX174 5.4 kb

• 1984

Epstein-Barr virus 170 kb

• 1995

Haemophilus influenzae 1.8 Mb

• 2001

Human 3 Gb

June 26th, 2000: working draft, 95% gesequenced April 14th, 2003: finished, 99% gesequenced Costs: $ 2.7 billion (instead of $ 3 billion) Timing: 1990 - 2003 (instead of 2005)

Bill Clinton Tony Blair Craig Venter Francis Collins

The Human Genome Project

• First Generation: a bit of history
• Next (Second) Generation
• Third Generation

Next Generation: Illumina

Sequencing Workflow

DNA isolation Library preparation Sequencing Data analysis

Sequencing Workflow

DNA isolation Library preparation Sequencing Data analysis

Sequencing Workflow

DNA isolation Library preparation Sequencing Data analysis

Illumina sequencing

• fragment DNA
• clonal amplification
• n flowcell by bridgePCR
• sequencing-by-synthesis

Bridge amplification

Illumina sequencing

• fragment DNA
• clonal amplification
• n flowcell by bridgePCR
• sequencing-by-synthesis

Sequencing by synthesis

Sequencing by synthesis

Per Cycle Imaging

G A T C

Per Cycle Imaging

G good quality G poor quality

Per Cycle Base Calling

Phred Score Incorrect base Accuracy 10 1 in 10 90 % 20 1 in 100 99 % 30 1 in 1000 99.9 % 40 1 in 10000 99.99 % 50 1 in 100000 99.999 % 0 to 93  ASCII 33 to 126 = single character

Quality Scoring

@SEQ_ID GATTTGGGGTTCAAAGCAGTATCGATCAAATAGTAAATCCATTTGTTC +SEQ_ID !''*((((***+))%%%++)(%%%%).1***-+*''))**55CCF>>>

FASTQ File

T A C G G T A C T T G C A T A G A T T A C G G T A C T T G C A T A G C T Alignment or Mapping of Reads R E F E R E N C E G E N O M E (HG19)

chromosome + position + strand

sample.bam

Run QC and filtering

sample.bam

sample.bam

• both reads
• quality scores
• chromosome
• position
• quality flag
• duplicate flag
• off target flag

sortedBAM file

Coverage T A C G G T A C T T G C A T A G A T T A C G G T A C T T G C A T A G C T A C G G T A C T T G C A T A G G A T T A C G G T A C T T G C G G T A C T T G C A T A G C T T T A C G G T A C T T G C A T

5x coverage

Variant Calling T A C G G T G C T T G C A T A G A T T A C G G T A C T T G C A T A G C T A C G G T G C T T G C A T A G G A T T A C G G T G C T T G C G G T G C T T G C A T A G C T T T A C G G T G C T T G C A T

G = homozygous alternative

G A T T A C G G T G C C G G T G C T T G C A T A G C T G C A T A G C T - A T T A C G G T G C T T G C A

Variant Calling T A C G G T G C T T G C A T A G A T T A C G G T A C T T G C A T A G C T A C G G T G C T T G C A T A G G A T T A C G G T A C T T G C G G T G C T T G C A T A G C T T T A C G G T A C T T G C A T

A/G = heterozygous

G A T T A C G G T A C C G G T G C T T G C A T A G C T G C A T A G C T - A T T A C G G T G C T T G C A

Variant Calling T A C G G T G C T T G C A T A G A T T A C G G T A C T T G C A T A G C T A C G G T G C T T G C A T A G G A T T A C G G T A C T T G C

A/G = heterozygous?

Variant Calling T A C G G T G C T T G C A T A G A T T A C G G T A C T T G C A T A G C T A C G G T G C T T G C A T A G G A T T A C G G T A C T T G C

G

sequencing quality good poor

MiniSeq MiSeq NextSeq500 HiSeq2500 2 x 150 b 2 x 300 b 2 x 150 b 2 x 125 b 6.6 Gb 13 Gb 100 Gb 450/900 Gb 22M clusters 22M clusters 0.4B clusters 2B/4B clusters 1 day 3 days 1 day 6 days 50k$ 100k€ 250k€ 700k$ 4250 $/WG 3500 $/WG

Illumina: Normal flow cell technology

HiSeq4000 HiSeqX Five HiSeqX Ten NovaSeq6000 2 x 150 b 2 x 150 b 2 x 150 b 2 x 150 b 0.65/1.3 Tb 0.8/1.6 Tb 0.8/1.6 Tb 0.85/1.7 Tb 2/4 B clusters 2.5/5 B clusters 2.5/5 B clusters 2.8/5.6 B clusters 4 days 3 days 3 days 2 days 900k$ 5 x 1.2M$ 10 x 1M€ 1M€ 2500 $/WG 1500 $/WG 1000 $/WG 1200 $/WG

Illumina: Patterned flow cell technology

Illumina: Patterned flow cell technology

• Patterned flowcell
• Billions of nanowells
• Extreme high density
• No overlapping clusters
• Special polymerase?
• ExAmp clustering
• primer swaps

• First Generation: a bit of history
• Next (Second) Generation
• Third Generation

Next Generation: Roche 454

Roche 454

• fragment DNA
• clonal amplification
• n bead by emPCR
• load beads in

PicoTiterPlate

• sequencing-by-

synthesis

Ion Torrent

Ion Torrent

• fragment DNA
• clonal amplification
• n bead by emPCR
• load beads on chip
• sequencing-by-

synthesis

• First Generation: a bit of history
• Next (Second) Generation
• Third Generation

Third generation sequencing = single molecule sequencing

Third Generation: PacBio

• last week update: bought by Illumina

RS Sequal

SMRT technology

• Library prep
• Circular DNA
• SMRT cell

• no DNA amplification
• real-time imaging of

DNA polymerase

• sequencing-by-

synthesis

PacBio

SMRT technology

• >10kb reads
• 1 Gb output
• Better chemistry
• De novo assembly
• Haplotyping
• Variant calling

Posted February 10, 2014 The Genomics Resource Center University of Maryland http://www.igs.umaryland.edu

Oxford Nanopore

Oxford Nanopore

Oxford Nanopore

Oxford Nanopore

ACCCGTCCG

• 6 bases in pore
• 6x base calling
• Caller development  Community

Oxford Nanopore

• High error rate, but major improvement in 2017…