NEXT GENERATION
SEQUENCING(BIG DATA), IT AND
THE CLINICAL PRACTICE
Che Martin Ph.D | PMP
Katrina Fox-O’Malley
Bulent Oral
BIG DATA …WHAT IS IT REALLY?


Has been made more popular due to advances in
computing and technology
Data that is :
High volume
 Variable
 Complex
 Can be mined to extract valuable information

MODERN APPLICATIONS-INFORMATION
EQUALS IMPROVED EFFICIENCY & PROFIT

Google :


UPS


Search engine and Company/Product –client matching
ORION program uses data such as speed, direction, performance and
other operations to optimize their performance.
Clinical and Research
Research applications : identification of new regulatory elements in
pathogens
 Identification of new drug targets
 Clinical applications – Precision medicine

Sources :
Thomas H. Davenport and Jill Dyche, "Big Data in Big Companies," May 2013.
McKinsey Global Institute” Big data: The next frontier for innovation, competition, and productivity “
NEXT GENOME SEQUENCING AND
PRECISION MEDICINE

Precision Medicine
Match treatment and diagnosis to a persons
molecular profile.
 Advances in molecular biology, genomics and other
technologies allow:

The molecular/genetic characterization of patient’s cancer
 In some cases apply these results to treatment strategies
that target molecular basis of the particular patient’s
cancer.
 Involves diagnostic tests via one of which is NGS to obtain
molecular information about cancer.

NGS STEPS -BIG DATA- HOW DOES IT
WORK
Sample are subjected to targeted sequencing;
known cancer genes that well-characterized as
mutational hot spots
 Sequencer produces reads of sequence data ~1.5
GB wells and ~ .1 GB fastq files (contain reads)
per sample

Source : https://rdp.cme.msu.edu/tutorials/init_process/RDPtutorial_INITIAL-PROCESS.html
NGS STEPS -BIG DATA- HOW DOES IT
WORK
Reads are quality checked and clipped
 Clipped read are:

Aligned to reference genome via one of many
algorithms.
 Converted to produce a BAM file. ~.3GB (per
sample)

Saved for visualization as part of downstream
analysis.
 BAM is processed by Variant calling Software
(many different algorithms)

NGS STEPS -BIG DATA- HOW DOES IT
WORK


Variant; genetic differences from reference genome (“normal
expectation”) are identified and confirmed via visualization.
Information is saved in VCF files
Source : http://www.sustc-genome.org.cn/pgi/documentation.php
NGS STEPS -BIG DATA- HOW DOES IT
WORK


Bioinformaticians :
Design pipelines to parse and annotate identified
variants with information required for the
clinical workflow:



Specific mutation (peptide)
Identifying relevant clinical trials
Identifying published references
Design verification pipelines (some cases)
 Design a infrastructure and pipelines to format
this data to be received by clinical LIMS
software.

ADT
Reports
/Results
Patient
Registration
Storage
Orders
Report
Generation
Patient Report
Electronic Medical Record
VCF
Sequencer (Big Data)
Bioinformatics
Pipeline
Laboratory Information System
LABORATORY INFORMATION SYSTEM (LIS)
SPECIMEN TRACKING AND PROTOCOL
DOCUMENTATION
SHARING DATA WITH BIOINFORMATICS
PIPELINE
Export
Aliases
Import new data and
match with sample
aliases
TECHNOLOGIST REVIEW
CLINICAL REPORT SENT TO EMR – HL7/PDF
REPORT CONTENT: PATHOLOGY &
SPECIMEN DETAILS
Cancer Gene Panel 50 w/ Interp Targeted Next
Generation Sequencing
Collected: 3/20/2015 11:17 AM
Received: 3/30/2015 8:26 AM
Verified Date/Time:
Specimen Information:
Surgical Path No.: S15-8646
Specimen Type: Paraffin Embedded Tissue
Tumor Type:Metastatic lung carcinoma
Block No.: B1
Neoplastic Cell Content: 50%
Institution:
REPORT CONTENT: CLASSIFICATION OF
VARIANTS
Result: The following variants were detected in the patient's specimen:
Tier 1
Gene Variant:
None detected
Type of Mutation:
Cosmic ID:
Tier 2
Gene Variant:
KRAS, c.34G>T, p.G12C
Type of Mutation:
SNV
Cosmic ID:
COSM516
Variants in Tier 2 may be associated with clinical trials. Please check www.clinicaltrials.gov for details
Tier 3
Gene Variant:
TP53, c.830G>T, p.C277F
Type of Mutation:
SNV
Cosmic ID:
COSM10749
Classification of variants: Variants are classified based on current evidence for clinical actionability.
Tier 1 – Clinical utility has been demonstrated - Actionable / Clinically Relevant variants.
-Variants in genes with approved therapeutic implications in specified tumors.
-Variants with potential diagnostic/classification, prognostic implications.
Tier 2 – Clinical utility /actionability has been documented.
-Variants with approved therapeutic implications in a different tumor type.
-Novel variants in genes that have approved therapeutic implications.
-Variants associated with Clinical trials.
Tier 3 – Variants of Uncertain Significance (VUS)
-Variants may be associated with little or no established cancer risk.

DISCLAIMERS/REFERENCES/E-SIGNATURE
METHOD: DNA was extracted from macrodissected, paraffin-embedded tumor of
the patient using the QIAmp Kit (Qiagen, Valencia, CA). The extracted DNA was
amplified and subjected to Next Generation Sequencing (NGS) using the Ion
Ampliseq Cancer Panel hotspot v2 on the Ion Torrent Personal Genome Machine (Life
Technologies). The targeted gene panel is designed to detect mutations/variants in 50
key cancer-related genes. This test was validated for mutations/Single Nucleotide
Variants (SNV) in the BRAF, EGFR, KRAS and JAK2 genes. The limit of detection is
precise and reproducible at approximately 5% with approximately 400X coverage and
2.5% with 1000X coverage. The data obtained was analyzed with the Torrent Suite
Software v 4.2. DNA sequences used as references for this panel of genes can be
found at http://www.ncbi.nlm.nih.gov/refseq/rsg/. The mutation nomenclature is
based on the recommendations from the Human Genome Variation Society
http://www.hgvs.org/mutnomen/.
Limitations: This mutation panel is designed to detect targeted mutations only. The
50 genes covered are not all sequenced in their entirety. Mutations outside the 207
interrogated amplicons will not be detected. Variants of uncertain origin (germline
versus somatic origin) cannot be determined unequivocally.
REFERENCES:
Sequist LV et al., First-Line Gefitinib in Patients With Advanced Non–Small-Cell Lung Cancer
Harboring SomaticEGFR Mutations J. Clin. Oncol , 2008, 26, 2442-2449.
Verified Date/Time:04/06/15 3:02 PM
By: John Doe M.D. (Electronic Signature)