2. Basics of NGS
• Fragmentation of DNA into a Library of smaller fragments.
• Libraries are then sequenced to be duplicated.
• Bioinformatics piece together the small fragments to create a map
and reference it to the human genome.
3. Differences between methods
PCR
Detects aneuploidy
Detects gene defects
Detects mitotic errors
1-2 month of Preparation
Requires affected proband
Applicable to any case
* Karyomapping using BlueGnome
PCR +
aCGH
SNP
Next Gen
arrays* Sequencing
no
yes
no
yes
no
yes
yes
yes
yes
yes
no
yes
yes
yes
no
no
yes
no
yes
yes
yes
no
no
yes
4. Next Generation Sequencing (NGS)
Fragmentation Each region of the genome
sequenced multiple times
GTACCATAGGATACGACTTGCAGCGGCA
ATATTGCGTATA
Millions of short sequences produced
CAGCGGCAGATGATTCGGGGATATTG
AGGATACGACTTGCAGCGGCAGATGATT
TGCGTATAGG
Sequences are compared to the known human
CAGATGATTCGGGGATATTGCGTA
genome
ACCATAGGATACGACTTGCAGCGGC
TAGAGTACCATAGGATACGACTTGCAACGGCAGATGATTCGGGGATATTGCGTATAGGCTA
Known sequence (CFTR gene chromosome 7)
Mutations identified and amount of DNA (aneuploidy) revealed
7. PGD for aneuploidy and gene defects
using NGS: Method
• With WGA only <10% is sequenced
• Solution: enrich the sequences of interest prior to NGS. An
aliquot of the WGA product was used to amplify by PCR the CF
ΔF508 mutation site on a cell line
• The gene was sequenced with a x30 depth
• All cells were found to be euploid and homozygotic for ΔF508.
• Conclusion: useful for DIRECT mutation analysis and aneuploid
D Wells, KKaur, A Rico, J Grifo, S Anderson, J Sherlock, JC Taylor , S Munne (submitted) Rapid genetic analysis of single
cells using a next generation sequencing methodology: application to human embryos reveals aneuploidy and DNA
sequence mutations
8. PGD for aneuploidy and gene defects
using NGS: Results
Cystic fibrosis gene (CFTR) F508 mutation sequenced in a single blastomere
D Wells, KKaur, A Rico, J Grifo, S Anderson, J Sherlock, JC Taylor , S Munne (submitted) Rapid genetic analysis of single
cells using a next generation sequencing methodology: application to human embryos reveals aneuploidy and DNA
sequence mutations
9. •
38 blastocysts from 13 couples with structural chromosomal
abnormalities
•
Whole genome sequence by Illumina HiSeq2000
•
Results: 0.07x depth with average 5.5% genome coverage
•
26 (68%) blastocysts euploid, 6 (16%) aneuploid, 4 (11%)
unbalanced only, 2 (5%) unbalanced and aneuploid
•
Highly concordant with SNP array results
Yin et al (2013) Biol Reprod 88, 69
10. •
21 blastocysts from couples at risk of cystic fibrosis and one of
Walker-Warburg syndrome
•
Whole genome amplification was followed by targeted Taqman
amplification of mutation site, sequenced by Ion Torrent and 8
barcoded samples per chip
•
Real time qPCR used for 24 chromosome aneuploidy testing
•
17 (81%) blastocysts euploid, 4 (19%) aneuploid
•
100% concordance of mutation status with STR and minisequencing
Treff et al (2013) Fertility and Sterility 99, 1377-1384
11. PGD for aneuploidy and gene defects
using NGS: Results
• A homozygotic cell line for ΔF508 was used
• The gene was sequenced with a x30 depth
• All cells were found to be euploid and homozygotic for ΔF508.
• Conclusion: this method can be use for DIRECT mutation
analysis and aneuploid
• Other methods such as SNPs can only do haplotype analysis
D Wells, KKaur, A Rico, J Grifo, S Anderson, J Sherlock, JC Taylor , S Munne (submitted) Rapid genetic analysis of single
cells using a next generation sequencing methodology: application to human embryos reveals aneuploidy and DNA
sequence mutations
12. Summary Next Gene Sequencing
Current Advantages:
- Same resolution for chromosome abnormalities than aCGH
- Detection of mitochondrial DNA: potentially useful
- Simultaneous detection of aneuploidy and gene defects
Future advantages:
- Whole gene sequencing
13. Vendors of Next Generation Sequencing
Company
Models
Benchtop Chemistry
Roche (454)
GS FLX/GS
Junior
Yes
Illumina
MiSeq/HiSeq/Ge Yes
nome Analyzer
Sequencing by synthesis
Ion Torrent
PGM/Proton/SO
LiD 4
Yes
Semiconductor sequencing
PACBio
PACBIO RS II
No
SMRT technology
Pyrosequencing
14. Vendors of Next Generation Sequencing
or Equivalent
Company
Models
Benchtop
Chemistry
Oxford Nanopore
Technologies
GridION System/MinION
No
Nanopore Sensing
RainDance Technologies
ThunderStorm
System/RDT 1000
Yes
High-Throughput/LowMed Targeted Sequencing
Helicos (Bankrupt)
N/A
N/A
N/A
Complete Genomics
Proprietary Sequencing
N/A
Proprietary (Long
Fragment Reads?)
15. Ion Torrent for Aneuploidy Validation
• 10 single cells from cell lines with known aneuploidies
• 40 embryo cells (previously diagnosed using aCGH)
•
•
•
•
Calculated amount of sequence from each chromosome
50/50 (100%) of samples gave a result
48 separate aneuploidies detected
100% diagnostic accuracy (in a blinded experiment)
16. First baby born from NGS
First NGS baby:
David Levy
A collaboration of
Reprogenetics-US and
Reprogenetics-UK
(Dagan Wells) and Main
Line Fertility (Dr.
Glassner)
17. Helpful tools for NGS
• https://genohub.com/next-gen-sequencing-services/
Editor's Notes
Above is Illumina created pictureBarcode adapters contain sequences that are optimized to provide equal representation of all barcodes in a pool
Usinga different approach i.e. Using an initial targeted PCR to enrich the region in which the mutation is located Treff and colleagues have shown accurate mutation detation and 100% concordance with STR and minisequencing. However, for aneuploidy parallel analysis by real time qPCR was used.... So not clear that there is any advantage in using the sequencer as minisequencing is well established and accurate etc...
Pyrosequencing – taking a single strand of the DNA to be sequenced and then synthesizing its complementary strand enzymaticallySequencing by synthesis - massively parallel sequencing of short reads using solid phase sequencing by reversible terminatorsSemiconductor sequencing – millions of wells capture chemical information that is related to a nucleotide.SMRT Technology – Phospholinked nucleotides are introduced to the DNA strand – occurs in real time
Nanopore sensing – events that create characteristic disruption in current.
