Process Steps Overview

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www.roche-applied- science.com 1 Process Steps Overview Data output DNA Library Preparation Prepare single-stranded DNA library with adapters Ready for titration sequencing run** Sequencin g Quality filtere d bases emPCR sstDNA with adaptors attached to bead Clonally amplified sstDNA in emulsion sstDNA ready to sequence ne library provides enough DNA for thousands of sequencing runs. Only one titration is required for each sample. 8 h 7.5 h 3. Sequencing 2. emPCR 4.5 h 1. DNA Library Construction * gDNA

description

3. Sequencing. 2. emPCR. 1. DNA Library Construction *. 4.5 h. 8 h. 7.5 h. emPCR sstDNA with adaptors attached to bead Clonally amplified sstDNA in emulsion sstDNA ready to sequence. Sequencing Quality filtered bases. Process Steps Overview. gDNA. Data output. - PowerPoint PPT Presentation

Transcript of Process Steps Overview

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Process StepsOverview

Data output

DNA Library Preparation Prepare single-stranded DNA

library with adapters

Ready for titration sequencing run**

Sequencing

Quality filtered bases

emPCR

sstDNA with adaptors attached to bead

Clonally amplified sstDNA in emulsion

sstDNA ready to

sequence*One library provides enough DNA for thousands of sequencing runs.** Only one titration is required for each sample.

8 h 7.5 h

3. Sequencing

2. emPCR

4.5 h

1. DNA Library Construction *gDNA

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Process Steps1. DNA library Construction Overview

sstDNA library

gDNA

A/B fragments selected using streptavidin-biotin purification

Denaturation to select for sstDNA library with A/B adaptors

No cloning; no colony picking

Library is created from any dsDNA

Genome fragmentation by nebulization

Ligation of adapters A & B

Data output8 h 7.5 h

3. Sequencing

2. emPCR

4.5 h

1. DNA Library Construction *gDNA

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Nebulization shears double-stranded DNA into fragments ranging from 50 to 900 base pairs.

High-pressure nitrogen gas is used to force the sample into small droplets of liquid which shears the DNA.

Nebulization

Snap cap

Condenser tube

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Fragment Distribution Post Nebulization

AMPure bead purification used to remove small fragments (<250 bp) Nebulized, purified sample run on Agilent 2100 DNA 1000 or 7500

LabChip Mean size between 400 bp and 800 bp < 10% of material smaller than 300 bp

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End Repair Reaction

Dr. Gary Kaiser, PHD

DNA ends are made blunt and phosphorylated. 3’ overhanging ends are removed (exonuclease). 3’ recessed ends are extended (polymerase). 5’ phosphates are added (kinase).

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DNA Ends Adapted with Specific Sequences

Ligase + left and right adaptors

Left adaptor Right adaptor

Polished insert DNA Left (A) and right (B) adaptor oligonucleotides are ligated onto the pool of nebulized polished genomic DNA.

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“A” adaptor 44 bases long 20 base PCR primer component 20 base sequencing primer component 4 base key

“B” adaptor 44 bases long 20 base PCR primer component 20 base sequencing primer component 4 base key Biotin on 5’ end (green dot)

Both adaptors are blunt on one end and recessed on the other to ensure only the blunt ends ligate to the polished genomic fragments.

“A” adaptor

“B” adaptor

GS Adaptors

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AB fragments

AA fragments

BB fragments

BA fragments

4 types of products are generated during ligation.

AB and BA products are equivalent (50%).

Products are bound to streptavidin-coated magnetic particles.

Ligation Products

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Adapted fragments are purified on a solid support and single-stranded material is eluted as the final product.

A

B

Isolate AB fragments only.

Library is Rendered Single-stranded

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3. Non-biotinylated

strands are melted

off and recovered.

(only the AB strand

will be captured)

1. AB and BB strands bind to magnetic particles.

2. Strands are

filled.

AB Strands Purified as the Final Library

AA products (no biotin) are washed away, BA and BB strands remain attached to magentic beads.

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Final Library Distribution

Typical single-stranded profile on Agilent 2100 RNA Pico 6000 LabChip.

Average size is 400-800 bp. Quantitate using Ribogreen Assay and dilute for emPCR Titration of DNA fragments is suggested to optimize the input copy

number for sequencing.

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Final Library Quantitation

Avogadro’s number is 6.022 x 1023 (molecules/mole) 328.3 x 109 (grams/mole) is the ave. molecular weight of

nucleotides Sample Concentration obtained from:

Agilent Bioanlayzer or via flourometry using a RiboGreen Assay Average fragment length obtained from

Agilent Bioanalyzer ONLY

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Summary Genomic DNA library construction

Nebulization – Shear DNA into appropriate size fragments Small fragment removal – SPRI based removal of fragments smaller

than 300 bp. DNA end repair – Make ends of DNA blunt and phosphorylated. Adaptor ligation – Add specific ends for amplification and

sequencing. Fragment immobilization – Bind fragment to solid support. Nick repair – Strand displacement to make fragments double-

stranded. Single-strand DNA isolation (library) – Isolate sstDNA fragments. Quantitation – Estimate the number of molecules recovered.

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Process Steps2. Emulsion PCR

Clonally-amplified sstDNA attached to bead

sstDNA library

Anneal sstDNA to an excess of 28 µm DNA Capture beads

Emulsify DNA Capture beads and PCR reagents in water-in-oil microreactors

Break microreactors and enrich for DNA- positive beads

Clonal amplification occurs inside microreactors

8 h 7.5 h

3. Sequencing

2. emPCR

4.5 h

1. DNA Library Construction *

gDNAData output

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GS FLX Technology Emulsion   PCR

From DNA quantitation, calculate a single DNA molecule to bead ratio for each microreactor

Wash Capture Beads

Anneal one DNA molecule to each Capture bead

Add PCR reagents to DNA+Capture bead

Transfer sample to oil tube

Shake to emulsify

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GS FLX Technology Emulsion Formation

Emulsion Oil and PCR mix containing

Capture Beads are mixed using a Qiagen

Tissue lyser as a high speed shaker

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GS FLX Technology Emulsion PCR

Emulsion oil – Before and After After emulsions are created, dispense into PCR tubes/plates

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GS FLX Technology Emulsion PCR

All samples processed in parallel

“B” primer is attached to capture bead.

“A” primer (in solution) is biotinylated.

Microreactors are amplified simultaneously.

Amplified products are driven to solid support (Capture Bead).

Each capture bead will contain ~10 million clonal copies.DNA Capture

Beads

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A) Anneal Single-stranded template to DNA Capture Beads

B) Emulsify millions of beads in PCR reagents to form water-in-oil microreactors• Microreactor contains complete

amplification mix

C) Thermocycle

D) Break Microreactors

E) Enrich for DNA positive beads

GS FLX Technology

Emulsion PCR

Before PCR

After PCR

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GS FLX Technology Breaking the Emulsion

Load Emulsion into Syringe Pass Emulsion through

Filter (beads are retained) Wash Beads using filter Recover beads from filter

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GS FLX Technology Enrichment

Beads with amplified DNA have the biotinylated ”A” primer

Beads with DNA product are extracted using streptavidin coated,

magnetic Enrichment Beads

Approximately 10% of beads have bound product

Add Enrichment Beads

Ma

gn

et

Bead with Amplified DNA

Bead without Amplified DNA

Enrichment Bead

Purify Beads with Product

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Process Steps3a. Bead Deposition into PicoTiterPlate ™

Well diameter average for PicoTiterPlate is 44 µm

A single clonally amplified sstDNA bead is deposited per well.

A layer of packing and enzyme beads are deposited

Plate is loaded into instrument for sequencing

Packed PTPAmplified sstDNA library beads

8 h 7.5 h

3. Sequencing

2. emPCR

4.5 h

1. DNA Library Construction *gDNA Data

output

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GS FLX Technology Assembling the Bead Deposition Device

The PTP is placed into the bead deposition device (BDD) bottom, a gasket is applied, the BDD top is placed over top and clamped securely in place.

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GS FLX Technology Loading Gaskets for 70X75 PTP

~420K reads ~280K reads

~192K reads

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GS FLX Technology Loading Gaskets for 25X75 PTP

~70K reads ~48K reads

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GS FLX Technology Bead Deposition Procedure

Each chamber is filled with DNA beads, packing beads and enzyme

beads in 3 separate layers

Prewet with BB2 (spin)

DNA (no spin)

Pack + BIM + Recycled Pol (spin)

Enzyme (spin)10 min

10 min

10 min

5 min

Recover

supernatant

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GS FLX Technology Bead Deposition

Empty PicoTiterPlate ™

DNA beads are loaded into the wells of the PTP.

DNA beads packed into wells with surrounding beads and sequencing enzymes.

*A well diameter of 44 µm allows for only 1 bead per well*

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Process Steps3b. Sequencing

High Quality readsAmplified sstDNA library beads

DNA capture bead containing millions of copies of a single clonal fragment

4 nucleotides (TACG) flowed for >100 cycles

Chemiluminescent signal generation

Signal processing to determine base sequence and quality score

300 - 400,000 reads obtained in parallel on a large format PicoTiterPlate

8 h 7.5 h

3. Sequencing

2. emPCR

4.5 h

1. DNA Library Construction *gDNA Data

output

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GS FLX Technology Sequencing Instrument

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GS FLX Technology Sequencing-by-synthesis

Simultaneous sequencing of the entire genome in hundreds of thousands of picoliter-size wells.

Pyrophosphate signal generation upon complimentary nucleotide incorporation — dark otherwise.

•Polymerase adds nucleotide (dATP)

•Pyrophosphate is released (PPi)

•Sulfurylase creates ATP from PPi

•Luciferase hydrolyses ATP and uses luciferin to make light

DNA capture bead containing millions of copies of a single clonal fragment

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Sequencing By Synthesis

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

C TA

Repeated dNTP flow sequence:

GGTCAGTCAGTTTTCAG GAT CCCGATT

G CT A

Anneal Primer

Process continues until user-defined number of nucleotide flow cycles are completed.

GS FLX Technology Sequencing-by-synthesis

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SoftwareImage-processing Overview

• Raw data is processed from a series of individual images.

• Each well’s data is extracted, quantified, and normalized.

• Read data is converted into flowgrams.

T

AG

CT

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SoftwareSignal Processing

Metric and image viewing software

Signal output from a single well (flowgram)

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SoftwareFlowgrams and Base calling

Key sequence = TCAG for identifying wells and calibration Flow of individual bases (TCAG) is 100 times.

TTCTGCGAA

TACG

Base flowSignal strength

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SoftwareFlowgrams and Base calling

Signal strength is determined by homopolymer length.

Flow Order

1-mer

2-mer

3-mer

4-mer

TACG

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Image capture

Image processing

Signal processing

GS denovo Assembler (denovo)

GS runMapper

(re-sequencing)

Process StepsData Output

GS Amplicon Variant Analyzier

(amplicons)

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Process StepsOverview of KitsLibrary Preparation

GS DNA Library Preparation Kit

Emulsion PCR (emPCR) GS emPCR Kits I, II, and/or III

> 1 emPCR kit for 70X75 sequencing run

Sequencing GSFLX Sequencing Kits and PicoTiterPlate Kit

Data Analysis/Interpretation Signal Processing, Basecalling, Assembly and Mapping