1. Bulk RNA-Seq Analysis Using CLCGenomics Workbench
December 11, 2018
Ansuman Chattopadhyay, PhD
Asst Director, Molecular Biology information service
Health sciences library system
University of pittsburgh

2. Topics Brief introduction to RNA-Seq experiments Analyze RNA-seq data
Dexamethasone treatment on airway smooth muscle cells (Himes et al. PLos One 2014)
Download seq reads from EBI-ENA/NCBI SRA
Import reads to CLC Genomics Workbench
Align reads to Reference Genome
Estimate expressions in the gene level
Estimate expressions in the transcript isoform level
Statistical analysis of the differential expressed genes and transcripts
Create Heat Map, Volcano Plots, and Venn Diagram

3. Differential Gene Expressions
Raw Reads
Venn Diagram
Volcano Plot

4. Workshop Page

5. HSLS MolBio

6. NGS Software @ HSLS MolBio
NGS Analysis
Sanger Seq Analysis
Human , Mouse and Rat
NGS Analysis

7. RNA-Seq Software @ HSLS MolBio
Enrichment Analysis
Deferentially Expressed Genes
CLC Genomics
Work Bench
Ingenuity
Pathway Analysis
Functions
Diseases
Pathways
RNA-Seq
Reads
Key Pathway Advisor
Upstream Regulators
Any Organism
Volcano Plot
PCA Plot
Venn
Diagram
Heat Map
Illumina BaseSpace
Correlation Engine
Correlated Expression Studies
CLC BioMedical
Work Bench
Variant Detection
Ingenuity
Variant Analysis
Human, Mouse and Rat
Variant Annotation
and Prioritization
RNA-Seq Analysis
Down Stream Analysis

8. CLCGx 12
Genomics Workbench
BioMedical Workbench

9. Install Plugins

12. CLCbio Genomics Workbench
System Requirements
Windows Vista, Windows 7, Windows 8, Windows 10, Windows Server 2008, or Windows Server 2012
Mac OS X 10.7 or later.
Linux: Red Hat 5.0 or later. SUSE 10.2 or later. Fedora 6 or later.
8 GB RAM required
16 GB RAM recommended
1024 x 768 display required
1600 x 1200 display recommended
Intel or AMD CPU required
Minimum 10 GB free disc space in the tmp directory

13. CLC Genomics Workbench @pitt
Mike Barmada, PhD

14. CLCBio Genomics Workbench Server
- You can connect your CLC Genomics Workbench software to the core HTC cluster available to University of Pittsburgh researchers through the Center for Research Computing (CRC).
- This allows you to transparently migrate data from your workstation to the cluster, and run analyses on the cluster, which then run independently of your workstation (i.e. you can shutdown your machine and your analyses will continue unabated).

15. Center for Research computing (CRC)

16. Request access to CRC

17. CLC Genomics workbench
Ensure you have the most up-to-date version of the CLCbio Genomics Workbench (the software should tell you if there's a more recent version when you start it, or you can check on the CLCbio website)
If you have not already done so, request a user account/allocation on the Center for Research Computing (CRC) for HTC cluster by filling out the required information
If your computer is not connected to the Pitt network (e.g. you are working from home or on a trip), or you are working from a laptop that is connected to the Pitt wireless system, make sure you setup Pitt VPN, so that you can communicate with the CLC Bioserver on HTC cluster.
Start the CLC Genomics Workbench

18. Connect to CLC Server

19. Access to CRC-HTC Cluster – CLC Server
If you DO NOT HAVE CRC-HTC account:
Use the following for a limited access
UserID: hslsmolb
PW: library1#
Server host: clcbio.crc.pitt.edu
Server host: 7777
If you have CRC-HTC account
Use – pitt user name; pitt password
Server host: clcbio.crc.pitt.edu
Server host: 7777

21. Pre-analyzed Results

22. Bulk RNA-seq Study

24. NCBI SRA

25. NCBI SRA

26. NCBI SRA
Untreated Vs DEX

27. Bulk RNA-seq Basic Steps
convert to cDNA fragments
adaptors ligation
short seq reads
align reads to reference genome
Wang Z, Gerstein M, Snyder M.
RNA-Seq: a revolutionary tool for transcriptomics.
Nat Rev Genet Jan;10(1):57–63.

28. Create Folder in CRC-HTC Cluster1 2

29. Create Workshop Folder@ HTC-CLC Server1 2 3

30. Illumina 1,131,359 4,330,403 NGS Technologies AB SoLid 18,495 25,170
NCBI Seq Read Archive
Illumina 1,131,359 4,330,403
AB SoLid , ,170
Ion Torrent , ,855
PacBio , ,097
MinIon
Tutorial: Galaxy NGS101 – Overview of NGS Technologies;

31. Nature Reviews on NGS Technologies

32. Illumina Technology https://vimeo.com/121178846

33. STEP 1: Import Reads to CLC2

34. STEP 1: Import Reads to CLC3 4 5

35. Help : Import Illumina Reads

36. Contact CLCBio Support Team

37. FASTQ format

38. Results By CLC : Imported Illumina Reads
CLC_Server_Data
-- achattopadhyay
----AnsumanC
---- workshop_RNA_Seq_May2016
----- Reads

39. Results By CLC: Imported Illumina Reads

40. CLC SRA Download

41. EBI ENA

42. EBI-ENA

43. STEP 1: Import Reads to CLC; Download from NCBI SRA2

44. FASTQC Project

45. Phred Score
wikipedia

46. Step 2: Create Seq QC Report1 2

47. Results By CLC: Read QC Report

48. RNA Seq Questionnaire
What is the scientific objective of the RNA Seq experiment? How many classes will be compared?
Are only coding RNA (mRNA) or long non coding RNA, miRNA expected to be detected?
Did all the samples pass RNA quality checks before sequencing?
Are there biological replicates? If so how many?
What type of sequencing platform was used to sequence the reads? Illumina, Ion torrent, Solid
Where was the sequencing performed? Facility name and contact info
When was the sequencing performed? Year/date
Which RNA – extraction method was used in the experiment? Total RNA/ poly A/ rRNA depletion method and kit name and if possible, link to protocol
Whether the protocol is strand specific or not? Unstranded/ forward/reverse, kit name and if possible link to protocol
Whether the data is single end or paired end?
What is the expected read length?
Do the reads contain adapters? If adapters present, what type of adapters? Adapter sequence, if available, or link (usually can get this info from facility)
What are the experimental conditions to perform differential expression analysis?
Which organism and the reference genome to be used for analysis?

49. Read Seq Trimming

50. STEP 3: Create Metadata Table

51. Step4: Import Metadata

52. Step4: Import Metadata 2 1 3

53. Step4: Import Metadata

54. STEP 5: Read Mapping

55. Read Mapping
Wikipedia

56. Read Mapping
Ozsolak et al.
Nature Review Genetics

57. RNA-Seq vs. Microarrays
covers more dynamic range
allows to discover novel transcripts
able to detect SNPs
more costly ($300-$1000/sample) than Microarray ($100-$200/sample)
Generates times larger dataset than Microarray
uncompressed RNA-Seq raw files: >5GB
Microarray
RNA-Seq
Riki Kawaguchi’s Blog:
Zhao S, Fung-Leung WP, Bittner A, Ngo K, Liu X. Comparison of RNA-Seq and microarray in transcriptome profiling of activated T cells. PLoS ONE Jan 16;9(1):e78644.

58. Must Read
Cresko Lab, University of Oregon

59. Best Practices

60. RNA-seq Analysis Pipeline

61. Popular Software

62. STEP 5: Read Mapping 5

63. STEP 5: Reads Mapping 7

64. STEP 5: Reads Mapping 8

66. Reference Genome http://www.gencodegenes.org/releases/current.html

67. STEP 5: Read Mapping

68. STEP 5: Read Mapping 9

69. STEP 5: Reads Mapping 10

70. STEP 5: Reads Mapping 12 11

71. Expression Values

72. STEP 5: Reads Mapping

73. Normalization Methods

74. STEP 5: Reads Mapping 12
Click on Role

75. STEP 5: Reads Mapping 13

76. Results By CLC: Reads Mapping

77. STEP 5: Reads Mapping; Fusion Tracks14

78. STEP 5: Reads Mapping; Fusion Tracks

79. STEP 5: Reads Mapping; Gene expression Track

80. Step6: Create a PCA Plot

81. Step6: Create a PCA Plot

82. Step7: Differential Expressions

83. Step7: Differential Expressions

84. Step7: Differential Expressions; Dex vs Unt

85. GraphPad Statistics Guide :

86. Step7: Differential Expressions; Dex vs Unt Volcano Plot

87. Step8: Create a HeatMap

88. Step8: Create a HeatMap

89. Step8: Create a HeatMap

90. Step8: Create a HeatMap

91. Step7: Create a Venn Diagram

92. Step7: Create a Venn Diagram

93. Step7: Create a Venn Diagram

94. Create a Track

95. Step8: Create a Track
Track for CRISPLD2

96. Step8: Create a Track
Track for CRISPLD2

97. Step8: Create a Track

99. Normalization Methods

100. Downstream Analysis DEG Annotates differentially expressed genes from
an RNA-seq experiment, using the curated
public data from GEO

101. NextBio Research

102. Export Data from CLC

103. Find Correlated Gene Expression Studies from GEO

104. Find Correlated Gene Expression Studies from GEO

105. Ingenuity IPA Analysis

106. RNA Seq Questionnaire
What is the scientific objective of the RNA Seq experiment? How many classes will be compared?
Are only coding RNA (mRNA) or long non coding RNA, miRNA expected to be detected?
Did all the samples pass RNA quality checks before sequencing?
Are there biological replicates? If so how many?
What type of sequencing platform was used to sequence the reads? Illumina, Ion torrent, Solid
Where was the sequencing performed? Facility name and contact info
When was the sequencing performed? Year/date
Which RNA – extraction method was used in the experiment? Total RNA/ poly A/ rRNA depletion method and kit name and if possible, link to protocol
Whether the protocol is strand specific or not? Unstranded/ forward/reverse, kit name and if possible link to protocol
Whether the data is single end or paired end?
What is the expected read length?
Do the reads contain adapters? If adapters present, what type of adapters? Adapter sequence, if available, or link (usually can get this info from facility)
What are the experimental conditions to perform differential expression analysis?
Which organism and the reference genome to be used for analysis?

107. Thanks To…. HSLS Carrie Iwema David Leung Michael Sweezer CLCBio
Shawn Prince
Center for Simulation and Modeling
Kim F Wong
Mu Fangping