1. RNA-SEQ IN PPMI Whole-Blood samples
Kendalll
Craig, Keller, Cookson, Van Keuren-Jensen
May 3rd, 2018

2. Overview »PI: Cookson, Craig, Keller, Van Keuren-Jensen
»Background: Develop a comprehensive RNA resource from whole blood samples that can be easily accessed and utilized by the PD research community
»Methods: Whole Transcriptome RNA-Seq from PaxGene whole blood
»Results (brief with possible figures): Phase I will be released at the end of May
»Next steps: Complete and release Phase II and small RNA-Seq for all samples. Complete data portal for easy/accessible gene look up
»Relevance for PPMI: Resource for examining transcriptomic changes, their relationship to genetic and clinical variables, and their potential as PD biomarkers

3. RNA-SEQ: BEYOND TRANSCRIPT QUANTIFICATION
Pre-spliced, Spliced, Strandedness
Junctions/Isoforms
Alternative start-sites
Integrated to individual w/ DNA
SNPs
Allele specific expression
Non-sense mediated decay, eQTLs

4. STUDY FLOW 1 HudsonAlpha Institute for Biotechnology 2 TGen/USC 3
PPMI-INFO LONI
- Data Transferred to Basespace
- Limited LIMs Info
Data Generation
-Initial Quality Control
-Analysis Tables
-Pilot Portal For Querying
Quality Control, Analysis, Portal
-Data Portal,
-Production Data Management
-Coordination
Data Sharing/Unblinding
BCL / FASTQ
Folders
BAMs/FASTQs Tables
Pilot Study
Main Study
Long RNA
Comparison of 4 Different Assays on 48 Samples
Comparison across 2 sequencing platforms, and 3 sites
Analysis of Depth vs. Species Detection
Small RNA
Comparison of 2 Different Assays
Long RNA
NovaSeq Sequencing
200M Reads/100M Read Pairs
4,623 Samples
Kappa + NEB
27 Plates Completed, Topping off underway
Small RNA
Finishing QC for production

5. Samples Subjects Baseline 6 Months YR1 YR2 YR3 PD 426 285 365 361 360
196
179
184
171
166
Prodromal 60 61 56
SWEDD 64 53 57
LRRK2 Affected
118
135
108
LRRK2 unaffected 79
138 98 43
SNCA and GBA
200
Genetic Registry
417 86
Samples: Longitudinal 4,500+ Samples on 1,000+ Individuals

6. DATA AND ANALYSIS FLOW FASTQs BAMs Tables Initial Release
Alignment
STAR
Genome Build/Transcripts Mirror TopMed
RNA-SeQC
Differential Analysis
Multiple Comparisons
DESeq2
FASTQs
Each: ~10-12 Gbyte
Phase 1: Tbytes
BAMs
Phase 1: Tbytes
Tables
Each: 1-5 Mb
Study: <10 Gbytes
Counts Gene,Transcript
P-val, Fold Δ Gene,Transcript
BAM Files
Integrative Analysis
+VCFs from WGS
Variant Calling, QTLs
Stats Reads..
VCFs WGS DNA
FASTQ Files
Descriptive Analysis
Gene/Transcript quantification
Salmon
TPMs Gene,Transcript
Tables: Sufficient for many uses.
FASTQs/BAMs: Require shipping of hard-drives

7. PHASE 1 PROGRESS: Initial 2,047 Samples
Ave. Read Pairs: Mil
or 218 Mil Reads
Med. Read Pairs: 107 Mil.
Over 90M: 1,944/2,437 Samples

8. Beyond Coding

9. Principal Component Analysis By Plate
Quality Control Overview
Principal Component Analysis By Plate
Principal Component Analysis By % Percent Correct Strand ~ 30% of Variance

10. Data Exploration Portal
Interactive Querying of Study Data
Landing Page
Study Overview
Pilot Study
Quality Control
Study Detail
Gene Query
Allows exporting of CSV/XLS
Allows visualization and comparison to other databases such as GTEX
Crosslinked to Relevant Biology and Disease Relevance
Portable/Secure
Single public HTML, w/ JSON API
Integrates via Node.js to OAUTH SSO
MongoDB, BigQuery version
HTML5/JS, D3.js
Cross-platform Validated

11. Acknowledgements Ivo Violich Michelle Webb Eric Alsop, Ph.D.
Elizabeth Hutchins, Ph.D.
Karen Crawford, Ph.D.
Art Toga, Ph.D.
Shawn Levy, Ph.D. + Team