AFA®- Enabled Workflows: - PowerPoint Presentation

Slide1

AFA®- Enabled Workflows: From Specimen Lysis to NGS and LCMS Analysis

APAC 2019

www.covaris.com

Slide2

Sample Prep

The current bottleneck in High Throughput Genome Analysis

Slide3

NGS Workflow with current non-multiplexed Library Prep

Sample Prep

NGS Library Prep

Sequence Generation

Data Analysis

Slide4

New multiplexed Library Prep addresses Throughput but creates new Bottleneck

Sample Prep

NGS Library Prep

Sequence Generation

Data Analysis

The Bottleneck Today

 

Slide5

Adaptive-Focused Acoustics:Precisely tuned Energy for a variety of Molecular Biology Workflow Steps

Tissue Rehydration

Cell Lysis

Lysate Homogenization

Disassociation of Macromolecular Complexes

Contact-free Mixing of Reactions (including SPRI Clean-up)

DNA Shearing

Slide6

Adaptive Focused Acoustics (AFA) Technology

Microcentrifuge Tube

Unfocused Transducer

Non-contact

Microcentrifuge Tube

Focused Waveguide

Sample Contact

Focused, Non-contact

Temperature regulated

Thermal Control is Key

Bath Sonicator

Probe Sonicator

Covaris AFA

Acoustic energy induces cavitation

that allows biomolecules to be manipulated

Slide7

AFA Platforms = Automated and Reproducible Sample Prep

Automation - High-Throughput

M220

S220

ME220

E-Series

8-96 samples

High throughput laboratory automation

Revolution R230

LE220Rsc

8-96 samples

High throughput laboratory automation

On-Deck with Liquid handler

Covaris

AFA consumable family; small and large sample volume processing, wide energy-range

Covaris

fully automation compatible consumables (SBS), low sample volume processing

Slide8

8

AFA-TUBE TPX = AFA Based Consumable

Covaris

Consumable for High Throughput Automation

Specially designed polymer-based consumable

LE220Rsc and Revolution R230 compatible

PCR-tube morphology

AFA treatment requires no fiber/beadsRFID-enabled sample tracking, LIMS integration

Compatible with most liquid handlers and on-deck processing units (SBS specifications)

Slide9

Sample Prep Bottlenecks – Not standardized or Automation Friendly

Sample

[LYSIS]

[Extraction]

Purification

Conventional

Bead Beating/Vortex

Lyticases

Harsh Chemicals

Volume Transfer

Phase Separation

Vortex or Pipette Mixing

Volume Transfer

Filter-based/vacuum manifold

Pipette Mixing

Volume Transfer

Covaris

AFA

DNA Shearing

Slide10

Standardized, automated and simplified Workflows driven by AFA

Mild lysis buffer

Small Volumes – AFA-TUBE compatible

Automated Homogenization & Dissociation

Contact-free mixing

One-vessel Protocol

Direct to NGS Workflows

Automation Workflows, Consumables & Platforms

Standardized and Reproducible Sample Prep

+

AFA

Sample

[LYSIS]

[Extraction]

Purification

DNA Shearing

Slide11

Cell Lysis and ExtractionDNA and RNA

Lysis Buffer Choice is important

truPOP

: High Molecular Weight DNA

TLB : RNA, DNA (and protein)

Slide12

AFA-enhanced Lysis & Extraction of HMW DNA in the AFA-TUBE

High Molecular Weight DNA

Lysis Buffer =

truPOP

truPOP

facilitates AFA-enhanced cell lysis and extraction of high molecular weight DNA

compatible with prokaryotic as well as eukaryotic cells

developed for small volume use in combination with the AFA-TUBE

compatible with downstream enzymatic processing

https://covaris.com/kits-and-reagents/trupop/

 

Sample

+

truPOP

(30-60µl)

R230 AFA

Lyse and Homogenize

+ Magnetic Beads/ Bind Solution

Magnetic Separation

R230 AFA

Mix

Wash Buffer

AFA Mix

Elution Buffer

AFA Mix

& Shear (optional)

Repeat

Library-Ready DNA

LE220Rsc

96 AFA-TUBE TPX Plate

Full Automation

Slide13

NGS Library Workflow: Contact-free mixing during SPRI Clean-up

AFA-mediated mixing for bind, wash and elute during SPRI clean-up.

AFA-mediated mixing during enzymatic reactions, e.g., Adapter-ligation.

AFA Mixing

Pipette Mixing

https://covaris.com/wp-content/uploads/M020088.pdf

Slide14

truPOP

:

Automation for a wide Variety of Sample Types / HMW DNA Extraction Workflow

Sample

Cell count

Yield

Treatment Time

Buffer

Volume

Instrument

Y.

Lipolytica

Culture

~ 3 x 10

7

~ 1.16 µg

60 min/plate

30

m

l

LE220-plus

S. Cerevisiae Culture

~ 5 x 10

7

~ 0.72 µg

15 min/plate

30

m

l

LE220-plus

E. coli (gram -) Culture

~ 4 x 10

8

~ 1.98 µg

<3 min/plate

30

m

l

LE220-plus

L. monocytogenes (gram +) Culture

~ 4 x 10

8

~ 1.95 µg

<3 min/plate

30

m

l

LE220-plus

hgDNA

whole blood 30 µl

~1.7 x 10

5

~600 ng

7 min/plate

30

m

l

LE220-plus

S. epidermidis

(gram +) Colony

~ 1 x 10

7

~100 ng

<1 min/strip

30

m

l

ME220

S. cerevisiae Y.

lipolytica

E. coli L. monocytogenes

hgDNA

-Blood S. epidermidis

Most conventional kits rely on multiple up-front steps to homogenize sample.

Bead beating or

vortexing

for tough to lyse specimens complicates automation.

AFA +

truPOP

Lysates are compatible with most purification workflows

& fully automatable from sample to DNA in one vessel.

Slide15

Whole Blood Nucleic Acid: From Sample to HMW DNA or RNA in the same Plate

“96

truXTRAC

Blood DNA”

QIAamp

96 DNA Blood (Qiagen)

Blood DNA Isolation 96-Well (

Norgen

)

Number of Reagents

6

6

6

Reagent Volumes (96 samples)

6 ml - 20 ml

2 ml - 50 ml

2 ml - 60 ml

Reagent Reservoirs

1

2

2

Lysate Mixing Step

AFA

Vortexing

Vortex in Single Tube

Centrifuge

No

Yes

No

Magnet

Yes

No

Yes

Lysate Transfer Step

No

Yes

Yes

Incubator

Yes (56C)

Yes (70C)

No

Pipette Racks (a' 96)

2

4

2

Hands off Automation

Yes

No

No

Slide16

https://covaris.com/wp-content/uploads/ABRF2019-Poster_HMW-Extraction-Whole-Blood.pdf

Slide17

DNA Shearing during SPRI Clean-up: Normalize your DNA before Library Prep

 

Sample

+

truPOP

(30-60µl)

AFA

Lyse and Homogenize

+ Magnetic Beads/ Bind Solution

Magnetic Separation

AFA

Mix

Wash Buffer

AFA Mix

Elution Buffer

AFA Mix

& Shear (optional)

Repeat

Library-Ready DNA

LE220Rsc

96 AFA-TUBE TPX Plate

Full Automation

Pulsing Repeats/

Iterations

Av. Mode

SD

%CV

10

591

29.3

5

30

313

9.3

3

60

182

10.6

5.8

Slide18

AFA-enhanced Lysis & Extraction of High Quality total RNA in the AFA-TUBE

RNA Lysis Buffer = TLB (Tissue Lysis Buffer)

TLB facilitates AFA-enhanced cell lysis and extraction of total RNA

compatible with prokaryotic as well as eukaryotic cells

developed for small volume use in combination with the AFA-TUBE

purification workflow essentially follows the one of HMW DNA extraction

 

Sample

TLB

(30-60µl)

AFA

Lyse and Homogenize

+ Magnetic Beads/ Bind Solution

Magnetic Separation

AFA

Mix

Wash Buffer

AFA Mix

Elution Buffer

AFA Mix

& Shear (optional)

Repeat

High Quality RNA

LE220Rsc

96 AFA-TUBE TPX Plate

Full Automation

Slide19

RNA from Bacteria:

E. coli

Cell Lysis & total RNA Extraction

AFA-TUBE-TPX/LE220-Plus; bath temperature 12°C; n x 2 second pulsed AFA-treatment.

Input 1-2 x 10

8

cells from liquid culture (LB) in stationary phaseLysis Buffer: TLBPurification: truXTRAC FFPE RNA purification reagents (Covaris)

Slide20

Lysis of Human Leukemia Cells and Extraction of High Quality total RNA

1 x 10

5

cells concentrated by brief centrifugation.

After media was removed, TLB (30µl) was added and lysis was induced by AFA.

Yield: 1.4 (+/-0.2) µg

DV200 >95%RIN >9Next:Determine amount of full-length (5’ mG capped).

Slide21

Total RNA extracted and purified from human Blood collected into

PAXgene

BCT

AFA –enhanced Lysis

Classical Vortex

Lysis

Yield (ng by Qubit) [CV]

191 [3.3%]

192 [1.9%]CV (%)3.31.9RIN Score7.8

5.7

CV (%)

10.3

17.5

DV200

84

73

CV (%)10.47.0

Q-Ratio* (200/71)0.670.56

* As described in:

Kashofer

et al. (2013)

PLoS

ONE 8(7): e70714.

Slide22

Cell Lysis and Co-Extractionof RNA and Protein

Lysis Buffer Choice is important

TLB : RNA & Protein

Extract from FFPE

Extract from fresh/frozen Cells (e.g., FNA)

Slide23

Total RNA and Protein extracted and from FFPE Tissue

Protocol developed for Broad Institute: LMD tissue sections from deparaffinized FFPE slides.

Essentially a modified FFPE (

tNA

) workflow.

TLB without Proteinase K (protein!) as lysis buffer.RNA is extracted and purified in the supernatant.

Protein is extracted from remaining tissue pellet.

Slide24

AFA-Enhanced Workflows: Eliminate Bottlenecks

AFA

Bacteria

Sputum

FFPE

Cells/Tissue

Microbiome

Blood

Liquid

Biopsy

Yeast

Sample Prep direct to NGS & PCR

No purification required (

truPOP

)

truXTRAC

Extraction & Purification

FFPE,

cfDNA

,

ChIP

Agnostic Workflows

Wide Range of Samples/Species

Adaptable Lysis Conditions

High Throughput Automation

No bead-beating, one-vessel lysis, extraction and purification

Slide25

So, what does this all mean?

Covaris

R&D is focusing on Biomolecule Extraction (RNA/DNA & Protein)

AFA-TUBE Configuration

Small Sample size; small lysis volume

Lysis, extraction and purification in ONE AFA-TUBE

Purified DNA and RNA are ready for NGS, PCR…Protein Extraction pursued in Collaboration (focus on Tryptic Digest LC-MS applications)

Co-extraction of RNA/DNA, RNA/Protein (future DNA/RNA/Protein)