Post on 12-Apr-2018
transcript
©2017 Oxford Nanopore Technologies. All rights reserved.
Rapid genomic screening of embryos using nanopore sequencing
Forman EJ & Scott RT Jr Contemporary OB/GYN ( 2014)
Euploid single-embryo transfer: the new IVF paradigm?
Daniel J Turner, PhD
Senior Director of Applications
Oxford Nanopore Technologies
©2017 Oxford Nanopore Technologies. All rights reserved.
Deamer’s notebook
Membrane
-
+
Nanopore
Translocation ~10 nm
• Protein nanopores occur naturally in cells and can embed themselves in membranes
• Nanopores create tiny holes or channels a few nanometers in diameter
• Molecules can pass from one side of a membrane to the other
Deamer & Branton et al., ~20 years ago
What is a nanopore?
©2017 Oxford Nanopore Technologies. All rights reserved.
Nanopore translocation
•Open current
•Red = current change 1
•Purple = current change 2
-
+
How does nanopore sequencing work?
Axopatch 200B - single channel
patch clamp amplifier
©2017 Oxford Nanopore Technologies. All rights reserved.
Nanopore translocation
•Open current
•Red = current change 1
•Purple = current change 2
-
+
How does nanopore sequencing work?
RNA ‘squiggle ’
Time (sec)
0 5 10 15 20 25
Curr
ent
(pA
)
50
100
150
200
250
open pore current open pore current
~1,500 nt transcript adapter polyA tail
©2017 Oxford Nanopore Technologies. All rights reserved.
Motor
Nanopore
Membrane
Connector flow
cell connects with
MinION
USB powers
device and passes
data to PC
Consumable
flow cell Sample port
Biological
nanopore
Array of
channels
Sensing
channel
Electronic
chip
Sensor chip: what’s inside:
How does nanopore sequencing work?
©2017 Oxford Nanopore Technologies. All rights reserved.
How does nanopore sequencing work?
1. Long reads: tens to hundreds of kilobases
2. Portability
3. Low cost
4. Data generation and analysis in real time
A. Run-until (no sample batching necessary)
B. Read-until
motor stall
tether oligo
motor protein
leader
strand
DNA of interest adapter adapter
©2017 Oxford Nanopore Technologies. All rights reserved.
Nanopore Applications
Environment
Cancer
research
Basic genome
research
Plant
research
Pathogens
Human
genetics
Transcriptomics
Population
genomics
Use-cases /
showcasing
Sample extraction &
library preparation
Microbiome
Clinical
research
Bioinformatics
©2017 Oxford Nanopore Technologies. All rights reserved.
Detecting aneuploidy with high-throughput sequencing
Euploid male Male with trisomy 13
Day 1 Day 3 Day 5 Biopsy of 1–3 cells gDNA extraction
Library preparation
and sequencing
Coverage normalised to haploid reference Coverage normalised to haploid reference
©2017 Oxford Nanopore Technologies. All rights reserved.
Why do PGS / PGD with nanopores?
Programme start:
diagnostic tests,
medical visits
3–4 weeks
Hormone
stimulation
7—9 days
Egg
collection
Blastocyst
biopsy
After 5 days
Embryo freezing /
vitrification
PGS test
~14 days
Embryo
thawing
Embryo
transfer
Medical supervision
over implantation
Patient’s next cycle
©2017 Oxford Nanopore Technologies. All rights reserved.
Why do PGS / PGD with nanopores?
1. Results faster
2. Lower cost per sample
3. Control over whole process
4. Lower startup cost
Programme start:
diagnostic tests,
medical visits
3–4 weeks
Hormone
stimulation
7—9 days
Egg
collection
Blastocyst
biopsy
After 5 days
Embryo freezing /
vitrification
PGS test
~14 days
Embryo
thawing
Embryo
transfer
Medical supervision
over implantation
Same cycle
©2017 Oxford Nanopore Technologies. All rights reserved.
ANXA5
• Placental anticoagulant
• M2 haplotype associated with recurrent miscarriage
• M2 parent —> expectant mother given heparin daily
• Testing embryo would stop unnecessary treatment
• PCR and nanopore sequencing identifies M2 haplotype
• Results confirmed by capillary sequencing
M2 SNVs
SNVs
deletion
insertion
short amplicon for WGA DNA
long amplicon for blood gDNA
ANXA5-003
ANXA5-001
exon 2 exon 1
Sample
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
A-C-C-A (M 2)
G-A-T-G (WT)
%of each h
aplo
type
0
20
40
60
80
100
© 2017 Oxford Nanopore Technologies. All rights reserved.
Combined aneuploidy screen and ANXA5 haplotyping
• WGA sample divided into 2
• One half used for ANXA5 haplotying assay
• One half used for aneuploidy screen
• Effective but inconvenient
Biopsy of 1–3 cells gDNA extraction Whole genome
amplification
End-prep of WGA DNA
Adapter and
tether attachment
Adapter and
tether attachment
End-prep of ANXA5 amplicon
PCR with specific primers
Sequencing
© 2017 Oxford Nanopore Technologies. All rights reserved.
Combined aneuploidy screen and ANXA5 haplotyping
• WGA sample divided into 2
• One half used for ANXA5 haplotying assay
• One half used for aneuploidy screen
• Effective but inconvenient
• Combined assay uses limited number of PCR cycles
• Sufficient WGA DNA is still present at the end of PCR
• All DNA is prepared for sequencing
• Low-coverage, whole-genome data and higher-
coverage amplicon data are generated together
• Easily adapted to other genes (e.g. Huntingtin)
Biopsy of 1–3 cells gDNA extraction Whole genome
amplification
PCR with specific primers
Simultaneous end-prep of ANXA5 amplicon
and accompanying WGA DNA
Adapter and tether attachment
Sequencing
© 2017 Oxford Nanopore Technologies. All rights reserved.
Combined aneuploidy screen and ANXA5 haplotyping
• All ANXA5 haplotypes confirmed by capillary
• All ploidy levels confirmed by aray-CGH
• Whole genome at low coverage for ploidy
• Specific region / regions at higher coverage for SNPs
• Long reads would allow amplicons of several kb
Sample: ONT34 Ploidy level
ANXA5 diplotype
47 , XX, +16
GATG (WT), homozygote Sample: ONT39
Ploidy level
ANXA5 diplotype
44 , XY, -14, -16
GATG (WT), ACCA (M2) heterozygote
Coverage normalised to haploid reference Coverage normalised to haploid reference
©2017 Oxford Nanopore Technologies. All rights reserved.
Coverage - how low can we go ?
• Ability to call aneuploidies robustly from low coverage: 50,000 reads, 500 nt in length required per sample = ~ 0.01x
• Can either multiplex to get low cost per sample, or use ‘run-until’ to get results very quickly
• Cheaper 128-channel flowcells will be available imminently
Number of reads
5,000 10,000 50,000 100,000
Accura
cy
20
40
60
80
100
Bin
wid
th (
kb)
20,000
10,000
5,000
1,000
500
100
©2017 Oxford Nanopore Technologies. All rights reserved.
Next steps
• No significant increase in coverage required to detect sub-chromosomal changes
• Microdeletions and duplications are 1–5 Mb and can arise by de novo NAHR events during meiosis
• Consequences can be devastating
Increasing resolution
Accura
cy
20
40
60
80
100
Number of reads
5,000 10,000 50,000 100,000
Bin
wid
th (
kb)
20,000
10,000
5,000
1,000
500
100
10—20 kb,
~95% homology
Dosage-sensitive
gene / genes
Proximal
Proximal
Distal
Distal
Centromere
Wild-type
Deletion
Duplication
©2017 Oxford Nanopore Technologies. All rights reserved.
Acknowledgements
Care Fertility
Simon Fishel
Oxford Nanopore Technologies
Sissel Juul
Eoghan Harrington
https://nanoporetech.com/
Applications team
All ONT staff