1. Cancer Genomics Core Lab
Update on CCC next generation sequencing capabilities and precision medicine testing
Cancer Genomics Core Lab
Co-directors: Lance Miller and Greg Hawkins

2. What is Next Generation DNA Sequencing?
Next Generation (also called Next-Gen) DNA sequencing (NGS)
is a system of massive parallel DNA sequencing performed in silico
(on chips or glass plates).
Capability to sequence a complete human genome in 24 hrs
The output from a single sequencing run >1 X 1012 bases

3. Network and pathway analyses
NGS Applications
point mutations
CNVs
WGS
exome
targeted capture
InDels
Structural
variations
Functional effects
Network and pathway analyses
Integrative analyses
Differential
expression
Transcriptomics
RNA-Seq
Gene fusions
Clinical
applications
Alternative
splicing
RNA editing
DNA methylation
Epigenomics
BS-sequencing
ChIP-Seq
Transcription
Factor binding
Histone
modification
Adapted from Shyr and Liu Biological Procedures Online 2013, 15:4

4. Illumina NextSeq 500 (Installed Jan 2015)
Up to 400 million reads (high throughput)
Up to 130 million read (mid throughput)
Data format: base-space
fastq, fasta, BAM, VCF
1 base pair sequencing
Run times (11-29 hours)
Manual library construction
Read lengths: single 1 X 50 to 1 X 150 bp
paired up to 2 X 150 bp
Capacity: High throughput
1 genome (20-30 X coverage)
8 RNAseq (up to 50 SE million reads)
>9 exomes (>30X coverage)
Capacity: Mid-throughput
3 exomes (>30X coverage)
3 RNAseq (up to 40 million reads)
Data analysis
BaseSpace (individual accounts)
Off-line: GATK, BWA, Bowtie, Cufflinks
Tophat, (lot’s of others)

5. Flow cell output Jan 2016 - v2 chemistry Q30 >90%
million clusters
Gbases
>150 million clusters
>45 Gbases

6. NGS Library Preparation
NGS Workflow
NGS Library Preparation
Sequencing
de-multiplexing of NGS libraries
read trimming
QC metrics/data filtering
base calling
BaseSpace
(automated)
Primary Analysis
.fastq files (curated raw reads)
Secondary Analysis
sequence alignment to reference genome
QC metrics/data filtering
.bam files (aligned reads)
variant calling and filtering (SNPs and InDels)
variant annotation
sequencing depth and coverage calculations
final QC metrics
User
Implemented
Tertiary Analysis
.vcf files (variant report)
RNAseq analysis
ChIPseq analysis

7. BaseSpace Dashboard https://accounts.illumina.com
all users get 1 Tb cloud storage free
free applications for data analysis

8. BaseSpace Run Metrics

9. BaseSpace Run Charts

10. BaseSpace Apps

11. Current CGC Applications for NGS
RNAseq
whole transcriptome, targeted gene panels: mRNA/microRNA/lncRNA, splice variants, gene fusions, allele specific expression, genetic variants
ChIPseq
Microbiome
16s ribosomal RNA sequencing
whole bacterial genome
Exome Sequencing
identification of coding variations
human, mouse, primate (vervet)
Targeted Sequencing
custom single gene or panel of genes
Methylation Sequencing
whole genome (bisulfite) or methylation capture (Methyl binding protein)
Whole Genome Sequencing
Targeted Panels (MiSeq)
predesigned (example: Cancer)
custom designed

12. Illumina MiSeq DX (Summer 2016)
1-25 million reads (high throughput)
Data format: base-space
fastq, fasta, BAM, VCF
1 base pair sequencing
Run times (5-24 hours)
Manual library construction
Read lengths: single 1 X 50 to 1 X 150 bp
paired up to 2 X 150 bp
Capacity: High throughput
8-28 samples per run (clinical)
>96 samples research
Type of Samples
ChIP-seq
Low throughput RNAseq
Sequencing panels
CLIA Approved
Cystic Fibrosis
Custom assay kit
Data analysis
BaseSpace (individual accounts)
Off-line: GATK, BWA, Bowtie, Cufflinks
Tophat, (lot’s of others)

13. Cancer Screening Panels Available for Clinical Diagnosis
(Next Generation DNA Sequencing)
Not all screening panels have the same content
Life Technologies
Agilent
Illumina

14. Sciclone G3 NGS Workstation – Perkin Elmer
480 libraries per week
192 exome captures per week Flexibility to run 8-96 samples per run
Intuitive user interface
Support for multiple protocols and kits
Sequence capture,
mRNA-Seq,
ChIP-SeqSample
indexing
normalization
NIH S10 or NCBC instrument grant (2016)

15. Contact Information
Co-Directors: Lance Miller, Greg Hawkins
Staff: Lou Craddock, Wei Cui, Jamie Haywood
Analyst: Dr. Jeff Chou
Location: Hanes 4026/4028, NRC 311/312
Contact
Lab Phone Number:

16. Questions and Discussion