Proof of principle assessment of a Next Generation ...€¦ · Proof of principle assessment of a Next Generation Sequencing workflow for rapid newborn screening and Cystic Fibrosis

Proof of principle assessment of a Next Generation Sequencing workflow for rapid

newborn screening and Cystic Fibrosis testing

16th June 2019

Rebecca Thomas & Elizabeth SollarsSheffield Children’s NHS Foundation Trust

• Organised and supported as part of the NHS

• There are 16 screening labs testing 30,000 – 130,000 babies per year

• Screening is performed free at point of care to the family

• The process is governed by agreed national targets and the Newborn Screening Committee

Confidential and copyright © Sheffield Children’s NHS Foundation Trust, June 2018

Newborn screening in the UK

In England, newborns are screened for nine rare but serious health conditions using dried blood spots:

Sickle Cell Disease (SCD), Cystic Fibrosis (CF), Congenital Hypothyroidism (CHT)

Newborn screening disorders

Plus 6 inherited metabolic diseases:– Phenylketonuria (PKU)– Medium-Chain Acyl-CoA

Dehydrogenase Deficiency (MCADD)– Maple Syrup Urine Disease (MSUD)– Isovaleric Acidaemia (IVA)– Glutaric Aciduria Type 1 (GA1)– Homocystinuria (pyridoxine

unresponsive ; HCU)


Newborn screening disorders

Proof of Principle: Can a Next Generation Sequencing

protocol be optimised for a high throughput capacity and a fast

turnaround time?


1) For disorders where there is no biochemical marker

suitable for newborn screening

2) A test that allows sampling shortly after delivery

before the baby leaves hospital

Early Genetic Testing

The ChallengeBirth

Dried blood spot

+ve resultClinical intervention

Day

0

5

7


Rapid NGS from a dried blood spot


Semi automated workflow

• Panthera DBS puncher at Sheffield NBS Facility • Use 6mm punch head • Punching time: <60 minutes per 96-well barcoded plate


Punching

• Optimised using Healthy Control Venous Blood and Dried Blood Spot samples (VB and DBS)

• Biomek FXp robot program developed

• 3x 96-well plates in 90 minutes


DNA Extraction

• Biomek FXp and NXp robot• Saves on manual handling time• Improves consistency

between samples• Removes potential

sources of error


Semi-automated AmpliseqLibrary F

Semi-automated Ampliseq Library Preparation

Robot

outperforms

manual library

preparation in

% Reads on Target

(p<0.04*)


• Ion Chef: 15 min hands on time;

11h run time (overnight); 2x chips

per run

• 192 samples per run using 2x Ion

540 chips

• S5 Prime: <15 min hands on time;

3h run time per chip

– run time + analysis = 6.5hr

• High-throughput bioinformatics

analysis pipeline


Chip loading and sequencing

• Designed and validated targeted AmpliSeq panel• Five genes; current NBS disorders• Panel designed to exons +/-5bp• Good coverage, except for part of CFTR ex1 & TSHR ex10.

NBS2 panel coverage

Gene Associated Condition

CFTR Cystic Fibrosis

ACADM MCADD

HBB Sickle Cell Disease

PAH Phenylketonuria (PKU)

TSHR Congenital

Hypothyroidism


NGS for NBS: NBS2 Panel


DBS vs. VB DNA Sequencing quality

DBS vs. VB DNA Variant calling Concordance


In 33 paired samples:

96% mean concordance

98% in regions of >30X coverage

Quality metrics from high throughput runs

First high throughput run (1706413) Second high throughput run (1707666, two chips)

Third high throughput run (1803070). Note scale for mapped reads when comparing to other plots.

Fourth high throughput run (1805276)

1st S5 Prime high throughput run (1808110) 2nd S5 Prime high throughput run (1901515) 3rd high throughput run (1901513)


Bioinformatics Pipeline

Automatic

data export

from

TorrentSuite

Query LIMS

to get list of

samples

Realignment

using TMAP

Variant

calling using

TVC

Quality &

ROI filteringQC check

Two-click automated pipeline

In-house

Python Flask

web

application

Bioinformatics Pipeline

Pipeline

progress

and QC

check

Automatic

data export

from

TorrentSuite

Query LIMS

to get list of

samples

Realignment

using TMAP

Variant

calling using

TVC

Quality &

ROI filteringQC check

Includes:

• Sample reception

• Booking onto Lab’s Information Management System

(LIMS)

• DBS punching

• DBS extraction

• AmpliSeq library prep, chip loading

• Robot (Biomek) service contract

• Ion Chef & S5 Prime contract

Does not include:

• Trust overheads

• Clinical scientist analysis & reporting time

With

labour

Without labour

96 samples £69.19 £67.57

93 samples £71.14 £69.48* Based on current discounts with Thermo

Fisher


Costing model

• 2 x 96 samples would be punched per day meaning that ~ 1000

samples could be sequenced per week

• Doubling up on automation equipment would increase sample high

throughput capability


Current timeline for NBC for NGS

Cystic Fibrosis Screening Project

Currently CF screening involves a biochemical pre-screen, and then a 4 and

50 mutation SNP array (2 mutations = affected, 1 mutation = carrier)

Can we expand testing to identify more mutations while keeping

within constraints of newborn screening programs?

Considerations for proof-of-concept:

DNA extraction & sequence from Dried Bloodspot

Turnaround Time (TAT) of 3 working days (samples received

Thursday, reports due Monday) Lab work

Pipeline

Interpretation

Identify carriers of 4 most common mutations, but not any others

Mutations have severity scores – only identify “clinically affected”

cases

Hotspot variant calling for known mutations quicker

interpretation


Cystic Fibrosis Screening Project

Results

9 months running NGS panel

• 201 total samples

• 14 reported carriers (of 4 common

muts)

• 21 affected newborns (7 wouldn’t have

genetic diagnosis without NGS)

We can detect CNVs using split reads over

breakpoints

All samples reported on time – apart from one

whole run fail.

Patient CF status and QC check


Sheffield Project Team:

Lead Applicants: Ann Dalton and Anne Goodeve

Co-applicants: Darren Grafham, Jim Bonham, Mark Sharrard, Lindsay Weaver, Pamela Davies and Diana Johnson

Sheffield Team: Rebecca Thomas, Elizabeth Sollars, Jennifer Dawe, Peter Winship, Michaela Novodvorksa, Clare Bartlett, Julia Van Campen ,Antonio Milano, Sian Richards, Gerrard Peck, Matthew Parker, Natalie Groves, Sufin Yap and Richard Kirk

Clinical Research Facility including Ally Spooner, Kim Redfern, Esther Ludbrook, Rachel Harrison, Carole Chambers and Samya Armoush

This case study presents independent research supported by the Health Innovation Challenge Fund (HICF-R9-518). The views expressed are those of the author(s) and not necessarily those of the Department of Health or Wellcome

Trust.

Collaborating NHS trusts:

BCH, CMFT, GST, GOSH, UHB, NUH, UHL, UCLH, STH

Acknowledgements

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


Documents

Proof of principle assessment of a Next Generation ...€¦ · Proof of principle assessment of a Next Generation Sequencing workflow for rapid newborn screening and Cystic Fibrosis