Download PPT File

New sequencing technologies in
cancer research
Alla L Lapidus, Ph.D.
Associate Professor
Fox Chase Cancer Center
Content
• Sequencing technologies
• Their use in Cancer research
• What is available at FCCC
• Data quality
“REVOLUTIONARY GENOME SEQUENCING TECHNOLOGIES
THE $1000 GENOME”
(Department of Health and Human Services (DHHS))
2004 - develop novel technologies that will enable extremely
low-cost, high quality DNA sequencing
2009 - the cost to sequence an entire individual human
genome to be $1,000 by the end of 2009 and the time
required for sequencing less than one week
Evolution of sequencing
J. Craig Venter, Nature 464, 676-677 (1 April 2010)
Sanger sequencing approach vs next-generation (next-gen)
approaches
Advances:
Cheaper (!)
no cloning involved => no cloning bias
deeper coverage can be obtained
Challenges:
shorter reads => problems for de-novo assemblies
higher error rate (homopolymer related errors in case of 454 and/or IonTorrent)
GC-bias still remains
slow turn around
old bioinformatics tools can not be used and more computer memory
and storage space is needed
3-d generation:
Price continuing to go down
Error rate is lower (Illumina, SOLID)
Read lengths increased
Bioinformatics is improved but space remains an issue (Amazon cloud etc)
• DNA fragmentation
• Fragment end repair and end
modification (adaptor/linker ligation)
• Library enrichment
• Sequence
DNA amplification
NA
At Fox Chase
Illumina
Dense lawn
of primers
DNA fragment
primers
Duplicate reads
10x
Coverage (depth)
Read coverage
Reference
Clone coverage
Cost of the project = f (Turnaround + Read coverage)
Read coverage = f (Signal to noise ratio)
We need high enough coverage to eliminate errors and better
quality reads for lower project cost!
Ion Torrent (LifeTechnologies) – 100bp reads, cheap,
simple
PacBio – read length 1kb
The Personal Genome Machine has been designed The PacBio RS system conducts, monitors and
for research purposes, offering to charge an iPod
analyzes single molecule, realtime (SMRT™)
whilst it analyses DNA. It has an iPod dock on top…
sequencing reactions. The instrument features high
performance optics, automated liquid handling, and
The rig uses parallel semi-conductor sensors to
an environmental control center, all accessed
measure the hydrogen ions produced during DNA
through an intuitive touchscreen user interface.
replication in real-time (pH…).
It is the first machine to use this type of
semiconductor technology, while still using
traditional integrated fluids and micromachining to
translate the information in our DNA into digital
information that can be easily measured.
So far there are no details as to the price of the device
http://www.iontorrent.com/?s_kwcid=TC-12648-4977112303-p-652132722
http://www.pacificbiosciences.com/products/pacbio-rs-system
Nanopores for DNA sequencing
An attractive strategy for single-molecule DNA sequencing is to pass single-stranded DNA through a
nanopore in a graphene monolayer. Here, the rings of carbon atoms in the graphene are depicted as
hexagons, and the diameter of the nanopore is about 1.5 nm, corresponding to about 35 hexagonal units.
The strand is moving from top to bottom in an applied electric potential, and each of the four DNA bases is
shown in a different colour. The DNA could be sequenced by observing the flow of ions through the pore
(vertical yellow shading) and recording the distinctive fluctuations of ionic current caused by each type of
DNA base as it blocks the ionic flow. Alternatively, fluctuations in a transverse tunneling current (horizontal
yellow shading) carried through the graphene, and modulated by DNA passing through the pore, could be
measured; the crocodile clips represent electrical connections. One possible problem is that single-stranded
DNA can adhere to graphene, as shown. Three scientific papers now report that fluctuations of ionic current
can be measured when DNA passes through a graphene nanopore, although the resolution of the
measurements is currently insufficient to detect and identify individual bases.
http://www.nature.com/nature/journal/v467/n7312/images_article/467164a-f1.2.jpg
Summary:
- New sequencing technologies allow to sequence genomes faster, at a lower
cost and with significantly higher coverage
- They change VERY rapidly
- Different technologies produce data with different error rates and error profiles
-Read length is between 30bp and 500bp
-Run time is between 4 hours and 2 weeks
-As a result you end up with a tremendous amount of data that needs to be
sorted through and analyzed
Content
• Sequencing technologies
• Their use in Cancer research
• What is available at FCCC
• Data qaulity
Sequencing project
DNA/RNA
isolation
Library
construction
Automatic
sequencing
Production:
- quantity and quality of produced data
- data formatting
Data
Analysis
Post-production:
- read alignment
- PI driven analysis
Cancer is a disease of genome alterations.
Which alterations can be detected:
M. Meyerson, S.Gabriel, G.Getz, Nature Reviews Genetics 11, 685-696
Applications
►Whole genome re-sequencing
►Targeted sequencing (regions, genes, exomes)
►de novo sequencing
►Whole transcriptome sequencing
►miRNA discovery and profiling
►DNA Methylation
►Histone Modification
►DNA-protein interaction
Somatic mutations
Modified from: M. Meyerson, S.Gabriel, G.Getz, Nature Reviews Genetics 11, 685-696
Recent publications - 1
,
Recent publications - 2
Recent publications - 3
The International Cancer Genome Consortium (ICGC)
Complexities of cancer genomics
1. Cancer samples differ from the peripheral blood samples that are used for germline genome
analysis in their:
 quantity – for example, diagnostic biopsies from patients contain only few cells
 quality - DNA/RNA from cancer are often of lower quality (formalin-fixed and
paraffin-embedded => increased background mutation rate)
 purity - mix of cancer and normal genomes
2. Cancers themselves may be highly heterogeneous and composed of different clones that
have different genomes
3. “Cancer genomes are enormously diverse and complex. They vary substantially in their
sequence and structure compared to normal genomes and among themselves. To
paraphrase Leo Tolstoy's famous first line from Anna Karenina: normal human genomes
are all alike, but every cancer genome is abnormal in its own way.” – M.Meyerson,
S.Gabriel , G.Getz, Nature Reviews Genetics 11, 685-696 (October 2010)
4. To identify somatic alterations in cancer, comparison with matched normal DNA from the
same individual is essential.
5. Costly whole-genome sequencing (tumor/normal) is the best approach to discover the
full range of genomic alterations — including nucleotide substitutions, structural
rearrangements, and copy number alterations — using just this single approach.
Cost of computing vs sequencing cost
Moore’s law: computers double in
power roughly every two years—
an increase of more than 30 times
over the course of a decade, with
reductions in cost.
(Moore's law describes a long-term trend in
the history of computing hardware. The
number of transistors that can be placed
inexpensively on an integrated circuit has
doubled approximately every two years. The
trend has continued for more than half a
century and is not expected to stop until
2015 or later.)
A map of human genome variation
from population-scale sequencing
The 1000 Genomes Project Consortium*
2 8 O C T O B E R 2 0 1 0 | VO L 4 6 7 |
N AT U R E | 1 0 6 1
Production group
People in different
aspects of data
analysis
- wgs of 179 individuals from 4 populations
- 2 mother-father-child trio (high coverage)
- Exone-targeted sequencing of 697 individuals
Content
• Sequencing technologies
• Their use in Cancer research
• What is available at FCCC
• Data quality
Whole Exome sequencing (WES) at FCCC
1.
Exone capture for all or selected genes
2.
Exone/exome sequencing
3.
Production data QC (data quality, exome coverage)
4.
Analysis
- Read alignment
- Total exome SNPs and small Indels identification (detection and annotation)
- dbSNP/1000Genomes filter
- synonymous changes filter
- additional filters based on the scientific task
- manual inspection (with some limitations)
- list of candidate genes for PI’s validation
Content
• Sequencing technologies
• Their use in Cancer research
• What is available at FCCC
• Data quality
Homozygous SNPs and indel
Poor alignment
Missed SNP?
Haplotype sequencing
If someone has two disease-linked mutations within a single
gene, it's difficult to determine with current genome
sequencing methods if there is one genetic mistake on
the maternal copy and one on the paternal copy or if both
variations lie within the same copy of the gene.
In the former case, the person has two defective genes,
which are likely to cause health problems.
In the latter, the person has one good copy of the gene and
one bad copy. In many cases, having the good copy can
compensate for the defective one.
"You lose a lot of information if you look at things at a genotype level versus a
haplotype level." Nicholas Schork, Scripps Research Institute, Nature Biotechnology.
Ways to haplotype whole genomes
1. University of Washington - combined next-gen sequencing with large
insert cloning (fosmids) to achieve a sequenced genome with haplotype
information
2. Stanford University - used microfluidics technology in combination with
genotyping to obtain haplotype information at the single-cell level
1. Combination of old and new sequencing approaches
-made fosmid library from DNA from a
HapMap individual (female) of Indian
descent
- split the library into more than
100 different pools
- Barcoded pools
- shotgun-sequenced the libraries on
the Illumina Genome Analyzer to a
mean depth of 2.4-fold per haploid
clone.
-whole-genome resequencing to search
for variants.(Illumina HiSeq 2000 with
50 base paired-end reads, 15-fold
coverage.
-assembled data into haplotype blocks
of different length (>37kb)
- phased variants (Genomic phase, the
assignment of alleles to homologous
chromosomes)
J. O. Kitzman Nature Biotechnology (2010)
What was detected:
(a) Homozygous deletion (top), hemizygous deletion (middle) and inversion (bottom) with fosmid clone support. Deletion
calls were made using read depth and paired-read discordance. Inversions were called by paired-read discordance. SNPs
within hemizygous deletions appear as stretches of hemizygosity by whole-genome shotgun sequencing. Purple connections
indicate the additional support of strand discordance of read pairs spanning genomic DNA and the vector backbone.
(b) Novel contigs not present in the reference assembly (red) but detected among clone pool–derived reads (light blue,
purple, yellow) are anchored by searching for positions in the reference common to those pools but missing from most or all
other pools. This approach anchors 1,733 recently reported insertion sequences including contig GU268019.
2. Single-Cell Approach
Whole-genome molecular haplotyping of single cells
-
use microfluidic device to captured single cells
-
protease digestion to release the chromosomes
-
randomly separated the chromosomes into 48
regions
-
chromosomes were then individually amplified
and analyzed with PCR, so that two pools with
differing homologous chromosomes could be
created
-
each pool containing one haploid genome, was
genotyped using an Illumina SNP array and by
creating haplotype blocks the size of a full
chromosome.
H.C. Fan, J.Wang, A.Potanina, S.R.Quake, Nature Biotechnology, 2010
THANK YOU!