exploring illumina’s newest sequencing solutions · pdf filedan gheba sr. sequencing...

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AppliedGenomics

Exploring Illumina’s Newest Sequencing

Solutions

Dan Gheba

Sr. Sequencing Specialist

September 14th, 2016

dgheba@illumina.com

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HiSeq HD Systems

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2014: HiSeq XPopulation Scale Human Genome Sequencing

1.8T | 6B READS | PE150 | <3 DAYS

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Sequencing Power for Every Scale

Decreasing price per GB

Incr

easi

ng s

yste

m o

utpu

t & p

rice

HiSeq 25001000 Gb | 4B

2x125NextSeq120 Gb | 400M

2x150MiSeq15 Gb | 25M

2x300

HiSeq 3000750 Gb | 2.5B

2x150

HiSeq 40001500 Gb | 5B

2x150

HiSeq X Ten1800 Gb | 6B

2x150

HiSeq X Five1800 Gb | 6B

2x150

MiniSeq7.5 Gb | 25M

2x150

RNA-seqExomesWGSMethylation

ProkaryoteTargetedQC

WGS – Large Scale

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HiSeq 4000:Over 2x more data in 1/3 the time!

1.5 TB | 5B READS | PE1503.5 DAYS

1 TB | 4B READS | PE1256 DAYS600 GB | 3B READS |

PE10011 DAYS

V3 V4 HD

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What can you do with just ONE LANE of HiSeq 4000 data?

Drosophila130 Mb Genome

715X Coverage

Mouse2.7 Gb Genome

34X Coverage

Arabidopsis157 Mb Genome

590X Coverage

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Innovative Patterned Flow Cell Technology

Undefined featureRandom spacing

Defined featureOrdered spacing

Nanowell Substrate Billions of Ordered Wells

Defined feature size

Optimal cluster spacing

Increased cluster density

Faster, Simplified imaging

Patterned FC Non-patterned FC

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TruSeq® Nano

TruSeq® PCR-free

Nextera® XT

Nextera® Mate-Pair

TruSeq® Synthetic Long Read

Nextera® Rapid Capture Exome

Nextera® Rapid Capture Custom

TruSeq® Exome

TruSeq® Rapid Exome

TruSeq® Stranded mRNA

TruSeq® Stranded Total RNA

TruSeq® RNA Access

TruSeq® Small RNA

TruSeq® ChIP

TruSeq® DNA Methylation

DNA Targeted DNA RNA / Regulation

Supported Library Prep KitsOn HiSeq 3000 and 4000 Systems

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ATAC-seq on the Hiseq 4000

Data from Eric Chow, Ph.D., UCSF

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ATAC-seq on the Hiseq 4000

Data from Eric Chow, Ph.D., UCSF

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Robust performance of patterned flow cells over a broad range of input concentrations with quality libraries

Specification is 312 M reads per lane75% PF yields 368 M reads per lane

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Nextseq 500

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The NextSeq® 500 Delivers on Three Key Aspects

Flexibility Speed Simplicity

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System enhancements: Optics

Power of high throughput appsSize and affordability of a desktop sequencer.

6xMiSeq imaging

capability

6xMiSeq imaging

capability

1/3Size of a HiSeq1/3

Size of a HiSeq

1/3Capital costof a HiSeq

1/3Capital costof a HiSeq

6 parallel miniaturized, solid-state optics modules

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Two channel SBS uses 2 images

Builds template over 5 cycles

Clusters appearing in green only are T

Clusters appearing in red only are C

Clusters appearing in both images are A

Clusters not present in either green nor red are G

Cluster intensities are plotted and bases are called accordingly

Two Channel SBS – NextSeq 500

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Fast 2-channel SBS

24 h

18 h

2x75bp run

MiSeq v3 NextSeq 500

FASTER CHEMISTRY

HALF THE IMAGES

25%faster

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2 x 150bp 2 x 75bp 1 x 75bp

Exome | Transcriptome18 | HOURS

Gene Expression Profile12 | HOURS

Human Genome30 | HOURS

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High120 GIGABASES | PE150

400M | CLUSTERS

Mid40 GIGABASES | PE150

130M | CLUSTERS

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One System, Two Output Modes

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Flexible applications

High-OutputUp to 120 Gb

400M clusters PF1 x 75 bp, 2x75 bp, 2 x 150 bp

20GEX

profiles

30xgenome

6-12 exomes

RNA-Seq

Mid-OutputUp to 40 Gb

130M clusters PF2 x 75 bp to 2 x 150 bp

6-36panels

2-3exomes

2-4samplesRNA-Seq

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What about Panels?

RNA-seq/Exome25M PE75

ChIP-seq10M SR50

Amplicon2M PE150

MiniSeq $935 $400 $120

MiSeq $875 $350 $125

NextSeq $170 $35 $25

$1,500 run : 25M reads

$1,530 run : 25M reads

$1,650 run : 130M reads

5X

Enabling a lower price point for large amplicon panels

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SIMPLE LOAD AND GO REAGENTSSETUP TIME ON PAR WITH MISEQ

WasteBuffer

Chemistry

Flow Cell

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CARTRIDGE FORMAT | AUTOMATED WASH | SELF-CLEANING

Designed for Simplicity and Efficiency

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When do you Wash?

Cartridge reagents contain wash solution

Post-run, there will be an automated wash

Use within 2 weeks and NO wash is required

Focus less on washing, more on sequencing!

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What is the potential?

Specification is 400 M ReadsIn this dataset - 600 million PF reads

(Add the 4 lanes circled)

https://basespace.illumina.com/s/DvjfrjGRZh6E

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What is the potential? https://basespace.illumina.com/s/DvjfrjGRZh6E

Specification is >80% q30In this example - >90% q30

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What are researchers doing with their NextSeqs?

For Research Use Only. Not for use in diagnostic procedures.

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Performance

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Side by side data quality - Error rateHiSeq 2500 vs NextSeq 500

Data trimmed to 2x75

HiSeq 2500 NextSeq 500

0.5%

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TruSeq Amplicon on NextSeq and MiSeq SequencersExcellent somatic variant calling performance

R2

NextSeq: 98.6%MiSeq: 97.3%

• 9 replicates HorizonDx Quantitative Multiplex Reference Standard. FFPE samples prepared with TruSeq Custom Amplicon, 2x151bp• Contact an Illumina representative for access to the NextSeq data set• TruSeq Amplicon Cancer Panel, Analysis with TruSeq Amplicon BaseSpace App v1.1. Stated limit of detection = 5% variant frequency

MiSeq® v3 NextSeq v2

Yield (NextSeq data downsampled to 8Gb) 8.1Gb 39.4Gb

% >Q30 90.3 92.2

Passing SNVs (expected = 8, VF >5%) 8 8

v2 Chemistry

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TruSeq Whole-Genome SequencingHigh precision and recall across platforms

Platform/Application Depth%

AlignedSNV

PrecisionSNV

RecallIndel

PrecisionIndel

Recall

HiSeq 2500 v4WGS (NA12878) – 350bp 39.8x 94.0% 99.9% 89.8% 96.6% 83.6%

HiSeq XWGS (NA12878) – 350bp 39.6x 90.6% 99.9% 90.7% 95.8% 83.0%

HiSeq 4000WGS (NA12878) – 350bp 38.2x 92.8% 99.8% 90.9% 95.4% 81.0%

NextSeq 500 v2WGS (NA12878) – 350bp 32.0x 90.6% 99.8% 90.9% 95.2% 80.7%

NextSeq 500 v2WGS (NA12878) – 550bp 33.3x 89.4% 99.8% 90.0% 94.8% 82.0%

Data sources: Most Projects below available in BaseSpace Public Data.• HiSeq 2500 v4: TruSeq PCR Free (4 replicates of NA12878)• HiSeq X Ten: TruSeq Nano (4 replicates of NA12878)• HiSeq 4000: TruSeq Nano 350 (NA12878, 6plex)• NextSeq 500 v2: TruSeq Nano 350 (NA12878)• NextSeq 500 v2: TruSeq Nano 550 (NA12878)

Data above analyzed using BWA Whole-Genome Sequencing v1.0 and VCAT 2.0 BaseSpace apps

For Research Use Only. Not for use in diagnostic procedures.

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Gene-level FPKM Comparisons

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HiSeq 4000

NextSeq 500

HiSeq 2500

HiSeq 4000

NextSeq 500

HiSeq 2500

GA

PDH

CA

LR

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Variant Calling Assessment

Unique to HiSeq 4000 Unique to HiSeq 2500

Common on both platforms

Consistent results between HiSeq 4000

and HiSeq 2500 platforms

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HiSeq 4000 vs HiSeq 2500 Callability Across AT / GC Regions

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Callability in AT / GC Regions

HiSeq 2500 2x126

HiSeq 4000 2x126

Comparison of AT/GC rich regions of the genome

Both platforms offer excellent coverage

Improved coverage in C rich and G rich regions on HiSeq 4000 system

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Callability in Genomic Regions

Consistent performance across both platforms

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Callability in Genomic Regions

HiSeq 2500 2x126

HiSeq 4000 2x126

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SNP Variant Frequencies (dbSNP)

Variant Frequency Variant Frequency

HiSeq 4000 HiSeq 2500

Freq

uenc

y

Freq

uenc

y

• Similar distributions for both platforms• Peaks at 50% and 100% variant frequency as expected

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SNP Variant Frequencies (not in dbSNP)

Variant Frequency Variant Frequency

HiSeq 4000 HiSeq 2500

Freq

uenc

y

Freq

uenc

y

• Similar distributions for both platforms• Mostly low-frequency variants

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Economics

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PE100/125/150Genomes

PE50/75Exomes or RNA‐seq

HD $        21  $        29 

2500 v4 $        32  $        53 

2000 v3 $        48  $        71 

2500 RM $        55  $        82 

NextSeq500 $        35  $        44 

Comparison Matrix – Paired End - Per GB

1.5X 1.5X

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RNA-seq ThroughputAssumes 50M read per sample requirement

5x Days a week $330 per sample

160 Monthly

3x per Month $480 per sample

~180 monthlyOr

4x per month $400 per sample

~370 monthly

3x Days a week $220 per sample

1200 Monthly

$900,000*

Shorter run times = Smaller hit on throughput due to service*all based on list

price

$250,000*

GD1

Slide 41

GD1 Gheba, Dan, 9/2/2016

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Sample Prep Automation

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Neoprep: Automation for the everyday lab

Up to

16samplesper run

• Sequencing-ready libraries in ~7–11 hrs

• 30 minutes of hands-on time

Run time: ~10.5 hrs

Hands-on time: 30 min.

NeoPrep Stranded mRNA

Recover sequencing-ready libraries

Collect libraries

NeoPrep system

Prepare samples for loading

TotalRNA

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Thank You!

dgheba@illumina.com