Download pptx - NGx Sequencing 101-platforms

Shawn C. Baker, Ph.D.

Sequencing 101 – NGS Platforms

Overview

Review Major Applications

Review Major Platforms

Future Trends

Asking the Right Questions

Applications

Whole Genome Sequencing

30X coverage

90 Gb

Long reads

Paired-end

Mate pair

Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)

Exome Sequencing

100X+ coverage

5 Gb

Long reads


Small Genome Sequencing

1+ Gb

Long reads

Paired-end

multiplexing

Credit: Rocky Mountain Laboratories, NIAID, NIH

Credit: Graham Colm (Wikipedia)

Targeted DNA Sequencing

10+ Mb

multiplexing


Transcriptome Sequencing

Lots of reads

Long reads


RNA Profile Sequencing

Lots of reads

multiplexing


ChIP Sequencing

Lots of reads

multiplexing


Metagenome Sequencing

Lots of reads

Long reads

Paired-end

Applications

Platforms

Platforms

Platforms

Illumina – Chemistry

Library Prep

Amplification

Sequencing

Illumina – Performance Specs

HiSeq 2000/25

00

HiSeq 1000/15

00GAIIx

HiScanSQ

MiSeq

Gb/run 600/120 300/60 95 150 7

Run time 11d/27hrs

8.5d/27hrs

14d 8.5d 35hrs

Gb/day 55/120 35/60 ~7 18 ~7

Read length

2x100/150

2x100/150

2x150 2x100 2x250

# of single reads/run

3B/600M 1.5B/300M

320M 750M 15M

Instrument cost

$690k/$740k

$590k/$640k

~$300k ~$400k $125k

Run cost ~$23k ~$11k ~$17k ~$11k ~$1k

Illumina – Applications

HiSeq 2000/25

00

HiSeq 1000/15

00GAIIx

HiScanSQ

MiSeq

Whole Genome

Exome

Small Genome

Targeted

Transcriptome

RNA Profiling

ChIP-Seq

Metagenomics

Illumina – Future Developments

HiSeq 2000/25

00

HiSeq 1000/15

00GAIIx

HiScanSQ

MiSeq

Focus on desktop system Longer reads Push into diagnostics Chemistry A = fast reads? Chemistry B = long reads?

Platforms

Life: SOLiD – Chemistry

Library Prep

Amplification

Sequencing

Life: SOLiD – Performance Specs

SOLiD 5500xl

SOLiD 5500xl

W

SOLiD 5500

SOLiD 5500 W

Gb/run 95 240 48 120

Run time 6 days 10 days 6 days 10 days

Gb/day ~16 24 ~8 12

Read length

2X60 2X50 2X60 2X50


~800M 2.4B ~400M 1.2B

Instrument cost

$595k $70k upgrade

$349k $70k upgrade

Run cost ~$10k ~$5k ~$5k ~$2.5k

Life: SOLiD – Applications

SOLiD 5500xl(

W)

SOLiD 5500(W)

Whole Genome

Exome

Small Genome

Targeted

Transcriptome

RNA Profiling

ChIP-Seq

Metagenomics

Life: SOLiD – Future Developments

SOLiD 5500xl(

W)

SOLiD 5500(W)

Probably very little Main focus is on Ion Torrent technology

Platforms

Life: Ion Torrent – Chemistry

Library Prep

Amplification

Sequencing

Life: Ion Torrent – Performance Specs

PGM 314

PGM 316

PGM 318

Proton 1

Proton 2

Gb/run 10-40 Mb 100-400 Mb

1 Gb ~10 Gb ~100 Gb

Run time 2 hours 2 hours 2 hours ~4 hours ~4 hours

Gb/day ~120 Mb ~1.2 Gb ~3 Gb ~30 Gb ~ 300 Gb

Read length

200b 200b 200b 200b >200b


~0.6M ~3M ~5.5M ~82M ~330M

Instrument cost

$50k $50k $50k $149k $149k

Run cost $349 $549 $749 ~$1k ~$1k

Life: Ion Torrent – Applications

PGM 314

PGM 316 PGM 318 Proton 1 Proton 2

Whole Genome

Exome

Small Genome

Targeted

Transcriptome

RNA Profiling

ChIP-Seq

Metagenomics

Life: Ion Torrent – Future Developments

PGM 314

PGM 316 PGM 318 Proton 1 Proton 2

Focus on desktop systems Longer reads Increased chip densities New machine? Push into diagnostics

Platforms

454/Roche – Chemistry

Library Prep

Amplification

Sequencing

454/Roche – Performance Specs

GS FLX+

GS Jr.

Mb/run 700 35

Run time 23 hours 10 hours

Mb/day 700 35

Read length

Up to 1kb ~400b


1M 0.1M

Instrument cost

~$500k $125k

Run cost ~$6k ~$1k

454/Roche – Applications

GS FLX+

GS Jr.

Whole Genome

Exome

Small Genome

Targeted

Transcriptome

RNA Profiling

ChIP-Seq

Metagenomics

454/Roche – Future Developments

GS FLX+

GS Jr.

Probably very little Roche has signaled the desire for new technology

Platforms

Pacific Biosciences – Chemistry

Library Prep

Amplification

Sequencing

Pacific Biosciences – Performance Specs

PacBio RS

‘C2’

Mb/run 120

Run time 40 min

Gb/day ~1 Gb

Read length

3kb (avg)


~50k

Instrument cost

~$700k

Run cost $100

Pacific Biosciences – Applications

PacBio RS

Whole Genome

Exome

Small Genome

Targeted

Transcriptome

RNA Profiling

ChIP-Seq

Metagenomics

Pacific Biosciences – Future Developments

PacBio RS

Longer reads Reduced error rate Increased chip density Non-fluorescent detection?

Other Platforms…

More Information…

www.blueseq.com

Matching Applications with Platforms

Whole Genome Sequencing


ILMN HiSeq

ILMN MiSeq

SOLiD

Ion PGM

Ion Proton

454 GS FLX+

454 GS Jr.

PacBio RS

✔

✔

✔

✔

Exome Sequencing


ILMN HiSeq

ILMN MiSeq

SOLiD

Ion PGM

Ion Proton

454 GS FLX+

454 GS Jr.

PacBio RS

✔

✔

✔

✔

Small Genome Sequencing

Credit: Rocky Mountain Laboratories, NIAID, NIH

Credit: Graham Colm (Wikipedia)

ILMN HiSeq

ILMN MiSeq

SOLiD

Ion PGM

Ion Proton

454 GS FLX+

454 GS Jr.

PacBio RS

✔

✔

✔

✔

✔

✔

Targeted DNA Sequencing


ILMN HiSeq

ILMN MiSeq

SOLiD

Ion PGM

Ion Proton

454 GS FLX+

454 GS Jr.

PacBio RS

✔

✔

✔

✔

✔

✔

Transcriptome Sequencing


ILMN HiSeq

ILMN MiSeq

SOLiD

Ion PGM

Ion Proton

454 GS FLX+

454 GS Jr.

PacBio RS

✔

✔

✔

RNA Profile Sequencing


ILMN HiSeq

ILMN MiSeq

SOLiD

Ion PGM

Ion Proton

454 GS FLX+

454 GS Jr.

PacBio RS

✔

✔

✔

ChIP Sequencing


ILMN HiSeq

ILMN MiSeq

SOLiD

Ion PGM

Ion Proton

454 GS FLX+

454 GS Jr.

PacBio RS

✔

✔

Metagenome Sequencing

ILMN HiSeq

ILMN MiSeq

SOLiD

Ion PGM

Ion Proton

454 GS FLX+

454 GS Jr.

PacBio RS

✔

✔

✔

Major Trends

Major Trends

Desktop machines

Ease of use

Faster runs

Diagnostic/clinical use

Longer reads

Nanopore technologies



Before you buy a platform Do I need to buy a platform? What are my major applications? What are my colleagues using?

Before you start sequencing What am I trying to answer? How will I analyze the data? Where is my variation coming from? How many samples do I need?