1. GE3M25: Data Analysis, Class3
TCD, 23/11/2017
Karsten Hokamp, PhD
Genetics

2. NGS 1 Intro-duction Week 10 Week 11 NGS 2 QC, Trimming Week 12 NGS 3
GE3M11Exam
Week 10
Week 11
Python 1
Intro-duction
Python 2
Strings and Files
NGS 2
QC, Trimming
Week 12
Python 3
File I/O, Branching
Python 4
Modules,Lists, Sets
NGS 3
Project files
Week 13
Python 5
Dictiona-ries
Python 6 Regex, System
NGS 4
Peak Calling
Week 14

3. Marks for GE3M25
Python exam: 50%
2/3 data
handling
1/3 statistics
ChIP-Seq report: 50%

4. Class 3: Project Data Download project data Quality assessment
Read mapping
(Trimming)
Visualisation

5. Class 3: Project Data http://bioinf.gen.tcd.ie/GE3M25/project
Antimicrob. Agents Chemother. (2014)

6. Class 3: Project Data Background: Candida genus of yeasts
most common cause of fungal infections worldwide
commensal
opportunistic
azole-based antifungal drugs
(CC) Image: Y. Tambe
Microscopic image (200-fold magnification) of Candida albicans ATCC 10231, grown on cornmeal agar medium with 1% Tween80 (

7. Class 3: Project Data Background: Azoles
class of five-membered heterocyclic compounds
discovery of ketoconazole in 1980s
inhibits 14-alpha-demethylase
(CC) Image: David E. Volk

8. Class 3: Project Data Background: Candida glabrata
most common non-albicans candida
highest incidence of azole resistance
mostly due to mutations in PDR1
or cells w/o mitochondrial function
Candida glabrata x.
Image: Billy J. Carver

9. Class 3: Project Data
Question: What are the direct gene targets of PDR1?
 ChIP-Seq analysis of pdr1Δ strain with tagged-PDR1 construct and treated with ethidium bromide

10. Class 3: Project Data

11. Class 3: Project Data

12. Next Generation Sequencing - Applications
Xu F, Wang Q, Zhang F, Zhu Y, Gu Q, Wu L, Yang L, Yang X. Impact of Next-Generation Sequencing (NGS) technology on cardiovascular disease research. Cardiovasc Diagn Ther 2012;2(2):

13. ChIP-Seq Basics ChIP = Chromatin ImmunoPrecipitation
Source: Bio-Rad
= highly ordered packaging of DNA and histones together

14. Rosa, S.; Shaw, P. Insights into Chromatin Structure
and Dynamics in Plants. Biology 2013, 2,

15. ChIP-Seq Basics
Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein.

17. ChIP-seq controls: Input chromatin Immunoglogulin G antibodies
Knock-outs
ChIP without antibody
Remove peaks that are due to cross-reactivity of AB
Remove unspecific binding (background noise)

18. GE3M25 Project Step 1 Download data
Browse to bioinf.gen.tcd.ie/GE3M25/ngs
Click on data link (class 3):

19. GE3M25 Project Steps in this class: 1. Download FastQ data set
2. Quality control (FastQC)
3. Storage of FastQC report file
4. Read mapping (Bowtie2)
5. Generate indexed and sorted BAM file
6. Visualisation (IGV)
7. Removal of PCR duplicates
8. Store BAM and index files

20. GE3M25 Project Data
Paired-end Sequencing vs Single-end Sequencing

21. GE3M25 Project Data files: Paired-end compressed FastQ files
xxxxxxx_exp_1.fastq.bz2 xxxxxxx_exp_2.fastq.bz2
xxxxxxx_ctrl_1.fastq.bz2 xxxxxxx_ctrl_2.fastq.bz2
2. Genome sequence
ChIP'ed data
Control data

22. GE3M25 Project Steps in this class:
1. Download FastQ data set  4 files
2. Quality control (FastQC)  on each file individually
3. Storage of FastQC report file
4. Read mapping (Bowtie2)  in pairs
5. Generate indexed and sorted BAM file  2 sets
6. Visualisation (IGV)
7. Removal of PCR duplicates
8. Store BAM and index files
(exp and ctrl)

23. GE3M25 Project Step 2 Info for project report
Data details (# sequences, read length, etc.)
What type of Quality encoding used
(Phred+33 or Phred+ 64)
Comments on quality aspects
Highlight of potential issues
Discuss ways to clean up data

24. Conversion of quality score:
Quality Information
Conversion of quality score:

25. GE3M25 Project Step 3 Storage of FastQC report
1. Open HTML report in browser
2. Copy and paste information into a Word document or
Ctrl-click to copy images (or use Grab for screenshots)
3. Mail document to you
store on USB/Network
upload HTML file through

26. GE3M25 Project – Read Mapping
Build an index of the Genome:
Syntax:
bowtie2-build fasta_file index_name
e.g.
bowtie2-build ASM254v2.fa C_glabrata
This name to be used in mapping step!

27. GE3M25 Project – Read Mapping
Bowtie2 mapping:
Single-end data:
bowtie2 -U _exp_1_fastq.bz2 -x C_glabrata -p 4 > exp1.sam
2. Paired-end data:
bowtie2 -1 file1 -2 file2 -x C_glabrata -p 4 > exp.sam

28. GE3M25 Project – Read Mapping
Bowtie2 mapping:
Single-end data:
bowtie2 -U _exp_1_fastq.bz2 -x C_glabrata -p 4 > exp1.sam
2. Paired-end data:
bowtie2 -1 file1 -2 file2 -x C_glabrata -p 4 > exp.sam
e.g.:
bowtie _exp_1_fastq.bz _exp_2_fastq.bz2 -x C_glabrata -p 4 > exp.sam

29. GE3M25 Project – Sorting and Indexing
Change SAM to BAM format:
tools/samtools view -b exp.sam > exp.bam
2. Sort
tools/samtools sort exp.bam > exp_sorted.bam
3. Sorting with 4 threads for speed-up:
tools/samtools sort 4 exp.bam > exp_sorted.bam
4. Generate an index
tools/samtools index exp_sorted.bam
 produces a file with .bai ending

30. GE3M25 Project Optional steps in this class: Soft-trimming (Bowtie2)
Trimming for quality and adapters (trim_galore)
 Try to maximise number of uniquely mapped reads! 5. Comparison of results
6. Upload of most suitable BAM and index files

31. GE3M25 Project Steps in this class: 1. Download FastQ data set
2. Quality control (FastQC)
3. Storage of FastQC report file
4. Read mapping (Bowtie2)
5. Generate indexed and sorted BAM file
6. Visualisation (IGV)
7. Removal of PCR duplicates
8. Store BAM and index files

32. GE3M25 Project Step 5 Generate indexed and sorted BAM file
Sequence Alignment/Map Format
- Standard format for read mapping results
- Can be compressed to save space:
binary SAM  BAM format
- Can be indexed for random access
- samtools allow viewing and processing SAM data

33. GE3M25 Project Steps in this class: 1. Download FastQ data set
2. Quality control (FastQC)
3. Storage of FastQC report file
4. Read mapping (Bowtie2)
5. Generate indexed and sorted BAM file
6. Visualisation (IGV)
7. Removal of PCR duplicates
8. Store BAM and index files

34. GE3M25 Project Step 6
Visualisation with IGV (Integrated Genome Viewer)
Requires several data files:
Genome
BAM file
BAM index file

35. (Integrated Genome Browser)
start IGV
(Integrated Genome Browser)

37. load 'ASM254v2.fa' from Download directory as genome

38. load from file:
mapped reads (exp.bam)

39.  click and drag region to zoom in

41.  coverage
 individual reads

42. Exercises Clean your data via trimming
Run bowtie with different parameters
How do these steps affect the number of mapped reads?
How do they affect the peaks that you see in IGV?

43. GE3M25 Project Storage of BAM file
upload BAM and index files (XXX.bam.bai) through upload page!
bioinf.gen.tcd.ie/cgi-bin/GE3M25/upload.pl

44. Don't forget to log out!