Module 1: Introduction to ChIP Sequencing and Analysis

Lecture

Lab

Breakdown of Markdown File

• At the start of each step, the intention will declare.
• This is then follow by a code block

Code:

Like this! This is the main code to run for the step.

Additionally the code block will include a header to indicate what environment to the run code for example:
###Shell###
pwd

###R###
getwd()

• Explaining for commands will be broken down following code block
• pwd & getwd()
  • See your work directory
• Sprinkled throughout will also include comments

Note

Important points and considerations will also be raised as so.

Module 1. Alignment

Step 1: Setup

• It’s always good practice to organize your directories beforehand
• This avoids file conflicts and accidental data loss.
• We’ll be making a subdirectory for each major step.

Code:

###Shell###
mkdir -p ~/workspace/module123/BWA_index
mkdir -p ~/workspace/module123/alignments
mkdir -p ~/workspace/module123/fastqc
mkdir -p ~/workspace/module123/stats
mkdir -p ~/workspace/module123/peaks
mkdir -p ~/workspace/module123/bigBed
mkdir -p ~/workspace/module123/bigWig
mkdir -p ~/workspace/module123/resources
mkdir -p ~/workspace/module123/diffBind
mkdir -p ~/workspace/module123/edgeR
mkdir -p ~/workspace/module123/qc

• mkdir -p ~/workspace/module123/BWA_index
  • mkdir creates a directory
  • -p creates the “parent” if the initial parent directory did not exist
  • how could we simplfy the command?
  • mkdir -p ~/workspace/module123/{BWA_index,alignments,fastqc,stats,peaks,bigBed,bigWig,resources,diffBind,edgeR,qc}

Step 2A: Retrieve a reference genome

• We’ll need a reference genome to align our sequences to.
• A great resource is UCSC’s genome repository containing latest Hg38 and older hg19 human genome.
• For our tutorial, we’ll be working chr19 of Hg38/Grch38. This is to speed demonstration purposes.

Code:

###Shell###
wget https://hgdownload.soe.ucsc.edu/goldenPath/hg38/chromosomes/chr19.fa.gz -O ~/workspace/module123/BWA_index/chr19.fa.gz
gunzip ~/workspace/module123/BWA_index/chr19.fa.gz -c > ~/workspace/module123/BWA_index/chr19.fa

• Runtime : <1 minute

• wget https://hgdownload.soe.ucsc.edu/goldenPath/hg38/chromosomes/chr19.fa.gz -O ~/workspace/module123/BWA_index/chr19.fa.gz

  • Wget is a command to pull online files.
  • We’re instructing it to pull chr19.fa.gz from the example address.
  • -O ~/workspace/module123/BWA_index/chr19.fa.gz instructs where and what we would like to save our file as.
  • What were to happen if we ran the command without -O?
  • Not all systems carry/support wget, Curl is another useful alternative.

• gunzip ~/workspace/module123/BWA_index/chr19.fa.gz -c > ~/workspace/module123/BWA_index/chr19.fa

  • gzip is a data compression format useful for reducing storage impacts
  • gunzip is the opposite decompresses gzipped data
  • -c is a STDOUT redirect. Normally running gunzip file.gz will turn file.tsv.gz into file.tsv. Running -c allows us to save the contents elsewhere. We do this we can compare the size difference.
  • compare ls ~/workspace/module123/BWA_index/chr19.fa.gz -ilth vs ls ~/workspace/module123/BWA_index/chr19.fa -ilth and note the size difference

Note

Prior to any tool usage, it’s good practice to explore the tool by invoking the help command. This may vary depending on tool. This is important as the same -O flag could have differing functions in different tools.

Step 2B: Index the genome

• Ready the genome fasta file by creating databases allowing tools to quickly access different parts of the file
• Indexing is only required once per genome.
• That being said, because there are different versions of Hg38/GRCh38 (such as those with or without ALT contigs and EBV) each version would need their own indices.
• Additionally index files are typically program/tool specific.
• Consortias will also have available the reference genome and related resources. E.g. https://ewels.github.io/AWS-iGenomes/

Note

A newer version of BWA-mem does exist, however we’ll be using the older version due to a bug that requires significant memory for indexing.

Code:

###Shell###
mkdir -p ~/workspace/module123/BWA_index
bwa index ~/workspace/module123/BWA_index/chr19.fa > ~/workspace/module123/BWA_index/index.log

• Runtime : <1 minute
• bwa index ~/workspace/module123/BWA_index/chr19.fa
  • Indexing step creates a database to enable quick retrieve of sequences
  • take a look inside your folder ls ~/workspace/module123/BWA_index/
  • For the lab, we’re utilizing BWA for alignment. ### Step 3A: FastQC and interpretation
• Checking the quality of your FASTQs is sanity check step (garbage in garbage out)

Code:

###Shell###
fastq_input=~/CourseData/EPI_data/module123/fastq/MCF10A.Input.chr19.R1.fastq.gz
fastq_h3k4me3=~/CourseData/EPI_data/module123/fastq/MCF10A.H3K4me3.chr19.R1.fastq.gz
mkdir -p ~/workspace/module123/fastqc
fastqc -t 8 ${fastq_input} -o ~/workspace/module123/fastqc > ~/workspace/module123/fastqc/input.log 2>&1
fastqc -t 8 ${fastq_h3k4me3} -o ~/workspace/module123/fastqc > ~/workspace/module123/fastqc/h3k4me3.log 2>&1

• fastqc
  • -t 8 specifies the number of threads we want to program to utilize
  • -o specifies our output directory ### Step 3A: FastQC and interpretation
• Let’s take a look at our FASTQC results and compare

Code:

###Browser###
http://main.uhn-hpc.ca/module123/fastqc/MCF10A.Input.chr19.R1_fastqc.html
http://main.uhn-hpc.ca/module123/fastqc/MCF10A.H3K4me3.chr19.R1_fastqc.html

Note

The URLs link to the TA’s instance, make sure to replace the domain with your own.

• Observe the summary report, note the warnings and errors.
• we can also look at reports at https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
• Per base sequence quality
  • a representation of quality across all reads where the ideal is average,median,25th and 75th resides within the green good quality space
  • X-axis is the BP position in your read
  • Y-axis is the base quality score
  • yellow boxes represent 25th and 75th percentiles
  • whiskers represent 10th and 90th percentiles
  • read line represents median
  • blue line represents average
  • For illumina reads, the first couple of bases tend to be poor, quality normalizes for majority of the read and slowly worsens approaching end of read
  • possible remedies are to trim reads
• Per tile sequence quality
  • a representation of quality by tile (a discrete subdivision with the lane of a flowcell) where heat is bad
  • would indicate an issue with the flowcell
• Per sequence quality score
  • a distribution of mean read quality score where the peak and body should be as far right as possible
• Per base sequence content
  • The % of AGCT across reads according to read position
  • generally expect an equal/even except for the first couple of base pairs (due to sequencing calibration)
  • Certain assays would change this for example : Bisulphite-Sequencing (unmethylated Cs become Us) or amplicon-sequencing (fixed ratios)
  • look at H3K4me3, why is it so high in GC? Promoters!
• Per sequence GC content
  • a distrubtion of reads’ GC content, where warning or failures are issued based on deviation of 15% or 30%
  • A shift in distribution could mean sequencing bias or contamination
• Per base N content
  • B/C Ns are low confidence calls by the sequencer, we expect very few instances
• Sequence Length Distribution
  • With Illumina sequencing, expect uniform distribution
• Sequence duplication levels
  • Using the first 200,000 to find duplicates: expect most reads to unique (left side), spikes otherwise indicate contamination or PCR artifacts
• Overrepresented sequences
  • a table breakdown of the sequences found above in Sequence duplication levels
• Adapter Content
  • detects if commonly used adapters remain in read sequence

Step 4: Alignment

• Mapping the read pairs to a position of the reference genome

Code:

###Shell###
ref=~/workspace/module123/BWA_index/chr19.fa
read1=~/CourseData/EPI_data/module123/fastq/MCF10A.Input.chr19.R1.fastq.gz
read2=~/CourseData/EPI_data/module123/fastq/MCF10A.Input.chr19.R2.fastq.gz
sample=MCF10A_input_chr19
bwa mem -M -t 4 ${ref} ${read1} ${read2} 2>~/workspace/module123/alignments/${sample}.alignment.log | samtools view -hbS -o ~/workspace/module123/alignments/${sample}.bam

• run time ~2 min

• The command can be broken down into the following pseudo code ALIGNMENT | SAMtoBAM_conversion. The | operate streams the results from the first command into the second.

• bwa mem -M -t 4 ${ref} ${read1} ${read2} 2>~/workspace/module123/alignments/alignment.log

  • • | the pipe delimiter passes the results to the next step ACTION A | ACTION B | ACTION C (like an assembly line)
  • bwa mem while BWA has many alignment algorithms, mem is best suited for efficiently handling >70bp paired end reads.
  • -M alters flagging of chimeric reads. By default chimeric reads are flagged as SUPPLEMENTARY (partially mapping), the option instead turns them to SECONDARY (multi-mapping). Needed to GATK/PICARD support downstream
  • -t 4 resource specification
  • 2>~/workspace/module123/alignments/alignment.log data outputted occurs in two streams 1 (the data) and 2 (debug messages, warnings and status updates). We redirect 2 to a log file.
  • What do we see in the logs?

Note

Logs contain valuable information, helpful for debugging.

• samtools view -hbS -o ~/workspace/module123/alignments/${sample}.bam
  • samtools view a tool to read our SAM,BAM,CRAM files
  • -hbS include header, output as BAM, input is SAM
  • -o specify what we want to save our file as
  • take a look our new BAM file. Note the header and the body.
• What other important functions of samtools?
  • CRAM conversion
  • samtools quickcheck
  • samtools view header
  • samtools flags ### Step 5: Coordinate Sort
• Rearrange our alignments by coordinates

Code:

###Shell###
sample=MCF10A_input_chr19
samtools sort -@8 ~/workspace/module123/alignments/${sample}.bam -o ~/workspace/module123/alignments/${sample}.sorted.bam

• run time <1 min
• samtools sort sorts our reads by genome coordinates
• Observe the files before and after via samtools view | head
• How to sort by readname? #### Step 6: Duplicate Marking
• Identify and tag alignments that are duplicates

Code:

###Shell###
sample=MCF10A_input_chr19
java -jar /usr/local/picard.jar MarkDuplicates \
I=~/workspace/module123/alignments/${sample}.sorted.bam \
O=~/workspace/module123/alignments/${sample}.sorted.dup_marked.bam \
M=~/workspace/module123/alignments/${sample}.dup_marked.output.log \
ASSUME_SORTED=TRUE \
VALIDATION_STRINGENCY=LENIENT \
> ~/workspace/module123/alignments/${sample}.dup_marked.error.log

• java -jar /usr/local/picard.jar MarkDuplicates I=~/workspace/module123/alignments/${sample}.sorted.bam O=~/workspace/module123/alignments/${sample}.sorted.dup_marked.bam M=~/workspace/module123/alignments/${sample}.dup_marked.output.log ASSUME_SORTED=TRUE VALIDATION_STRINGENCY=LENIENT
  • java -jar /usr/local/picard.jar produced by BroadInstitute, Picard tools are a toolset for HTS/NGS data
  • MarkDuplicates identifies reads/clusters duplicates arising from library construciton during PCR and cluster formation during sequencing
  • I= and O= are input and output respectively
  • M= saves our output log
  • ASSUME_SORTED=TRUE informs PICARD, our input is already coordinate sorted
  • VALIDATION_STRINGENCY=LENIENT informs PICARD to continue and notify problems in the work.log instead of failing

Step 7: Stats

• Identify and tag alignments that are duplicates

Code:

###Shell###
sample=MCF10A_input_chr19
mkdir -p  ~/workspace/module123/stats
samtools flagstat ~/workspace/module123/alignments/${sample}.sorted.dup_marked.bam > ~/workspace/module123/stats/${sample}.sorted.dup_marked.flagstat
samtools stats ~/workspace/module123/alignments/${sample}.sorted.dup_marked.bam > ~/workspace/module123/stats/${sample}.sorted.dup_marked.stat

• samtools flagstat tallies FLAGS and produces a summarized report
• samtools stats collects various metrics on the BAM file include:
  • summary stats similar to flagstats
  • distribution of insert sizes
  • distribution of read lengths
  • distribution of Coverage
  • distribution of GC-depth
  • Includes detailed instructions on how to extract parts of interest

Step 8: Processing the remaining files

• we use a unix for loop to perform all the steps previously mentioned but for the other histone marks and ATAC-seq Code:

###Shell##
ref=~/workspace/module123/BWA_index/chr19.fa
for histone in H3K27ac H3K27me3 H3K4me3 ATAC;
    do
    read1=~/CourseData/EPI_data/module123/fastq/MCF10A.${histone}.chr19.R1.fastq.gz
    read2=~/CourseData/EPI_data/module123/fastq/MCF10A.${histone}.chr19.R2.fastq.gz
    sample=MCF10A_${histone}_chr19
    echo "aligning ${histone}"
    bwa mem -M -t 4 ${ref} ${read1} ${read2} 2> ~/workspace/module123/alignments/${sample}.alignment.log | samtools view -hbS -o ~/workspace/module123/alignments/${sample}.bam
    echo "sorting ${histone}"
    samtools sort -@8 ~/workspace/module123/alignments/${sample}.bam -o ~/workspace/module123/alignments/${sample}.sorted.bam
    echo "dupmarking ${histone}"
    java -jar /usr/local/picard.jar MarkDuplicates I=~/workspace/module123/alignments/${sample}.sorted.bam O=~/workspace/module123/alignments/${sample}.sorted.dup_marked.bam M=~/workspace/module123/alignments/${sample}.dup_marked.output.log ASSUME_SORTED=TRUE VALIDATION_STRINGENCY=LENIENT > ~/workspace/module123/alignments/${sample}.dup_marked.error.log
    echo "calculating stats ${histone}"
    samtools flagstat ~/workspace/module123/alignments/${sample}.sorted.dup_marked.bam > ~/workspace/module123/stats/${sample}.sorted.dup_marked.flagstat
    samtools stats ~/workspace/module123/alignments/${sample}.sorted.dup_marked.bam > ~/workspace/module123/stats/${sample}.sorted.dup_marked.stat
    done

• run time ~8 mins
• for loops are different in every language, best practice to review beforehand
• have feedback to indicate where in the process we are e.g. the echo lines
• not show here but also good to capture success and failures

Step 9: Clean up!

• Remove temporary files

Code:

###Shell###
ls ~/workspace/module123/alignments/*.bam | grep -v sorted.dup_marked | xargs -I {} sh -c "echo rm {}; rm {}"

• ls ~/workspace/module123/alignments/*.bam
  • a look up of BAM files in our directory : ~/workspace/module123/alignments
  • The * in *.bam acts as a wild card for any string of variable length ending in the suffix bam
  • running the command on it’s own produces
• grep -v sorted.dup_marked
  • grep a powerful tool that searches and matches text
  • -v return matches that do not much our pattern or regex
  • sorted.dup_marked is our pattern, returning 2/3 of the files : MCF10A_input_chr19.bam,MCF10A_input_chr19.sorted.bam,MCF10A_input_chr19.sorted.dup_marked.bam
• xargs -I {} sh -c "echo rm {}; rm {}"
  • xargs receives input and performs an action. Think a for-loop
  • -I {} the variable we want to use
  • sh -c interpret the string provided in bash shell
  • echo rm {} echos the command we want to perform
  • rm {} removes the file

Server resources

QC Resources

###TSS+/-2kb
mkdir workspace/module123/qc
wget https://www.bcgsc.ca/downloads/esu/touchdown/hg38v79_genes_tss_2000.bed -O workspace/module123/resources/hg38v79_genes_tss_2000.bed

sort -k1,1 -k2,2n workspace/module123/resources/hg38v79_genes_tss_2000.bed > tmp
mv tmp workspace/module123/resources/hg38v79_genes_tss_2000.bed

###Enhancer liftover
wget https://www.bcgsc.ca/downloads/esu/touchdown/encode_enhancers_liftover.bed -O workspace/module123/resources/encode_enhancers_liftover.bed

###Blacklist
wget https://www.encodeproject.org/files/ENCFF356LFX/@@download/ENCFF356LFX.bed.gz -O ~/workspace/module123/resources/hg38_blacklist.bed.gz

gunzip ~/workspace/module123/resources/hg38_blacklist.bed.gz

• hg38v79_genes_tss_2000.bed
  • Generated by downloading Ensemblv79 GTF convert to Bed +/-2kb of TSS. See https://www.biostars.org/p/56280/
• encode_enhancers_liftover.bed
  • download various ChroHMM state7 and merge
  • converted from hg19 to hg38 using UCSC liftover tool

Encode Bed

ls ~/CourseData/EPI_data/module123/encode_bed
basal.H3K27ac.peak_calls.bed
basal.H3K27me3.peak_calls.bed
basal.H3K4me1.peak_calls.bed
basal.H3K4me3.peak_calls.bed
lp.H3K27ac.peak_calls.bed
lp.H3K27me3.peak_calls.bed
lp.H3K4me1.peak_calls.bed
lp.H3K4me3.peak_calls.bed
luminal.H3K27ac.peak_calls.bed
luminal.H3K27me3.peak_calls.bed
luminal.H3K4me1.peak_calls.bed
luminal.H3K4me3.peak_calls.bed
stromal.H3K27ac.peak_calls.bed
stromal.H3K27me3.peak_calls.bed
stromal.H3K4me1.peak_calls.bed
stromal.H3K4me3.peak_calls.bed

• https://epigenomesportal.ca/tracks/CEEHRC/hg38/
• Breast Basal CEMT0035
• Breast Stromal CEMT0036
• Breast Luminal CEMT0037
• Breast Luminal Progenitor CEMT0038 #### Encode BigWig

ls ~/CourseData/EPI_data/module123/encode_bigWig
basal.H3K27ac.signal_unstranded.bigWig
basal.H3K27me3.signal_unstranded.bigWig
basal.H3K4me1.signal_unstranded.bigWig
basal.H3K4me3.signal_unstranded.bigWig
lp.H3K27ac.signal_unstranded.bigWig
lp.H3K27me3.signal_unstranded.bigWig
lp.H3K4me1.signal_unstranded.bigWig
lp.H3K4me3.signal_unstranded.bigWig
luminal.H3K27ac.signal_unstranded.bigWig
luminal.H3K27me3.signal_unstranded.bigWig
luminal.H3K4me1.signal_unstranded.bigWig
luminal.H3K4me3.signal_unstranded.bigWig
stromal.H3K27ac.signal_unstranded.bigWig
stromal.H3K27me3.signal_unstranded.bigWig
stromal.H3K4me1.signal_unstranded.bigWig
stromal.H3K4me3.signal_unstranded.bigWig

• https://epigenomesportal.ca/tracks/CEEHRC/hg38/
• Breast Basal CEMT0035
• Breast Stromal CEMT0036
• Breast Luminal CEMT0037
• Breast Luminal Progenitor CEMT0038 #### MCF10A Fastq

ls ~/CourseData/EPI_data/module123/fastq
MCF10A.ATAC.chr19.R1.fastq.gz
MCF10A.ATAC.chr19.R2.fastq.gz
MCF10A.H3K27ac.chr19.R1.fastq.gz
MCF10A.H3K27ac.chr19.R2.fastq.gz
MCF10A.H3K27me3.chr19.R1.fastq.gz
MCF10A.H3K27me3.chr19.R2.fastq.gz
MCF10A.H3K4me3.chr19.R1.fastq.gz
MCF10A.H3K4me3.chr19.R2.fastq.gz
MCF10A.Input.chr19.R1.fastq.gz
MCF10A.Input.chr19.R2.fastq.gz

• MCF10A histone marks and input come courtesy of Dr.Hirst
• ATACseq data originates from GSM6431322 #### Triplicates

CourseData/EPI_data/module123/triplicates/triplicates.csv

CourseData/EPI_data/module123/triplicates/alignments:
MCF10A_H3K4me3_chr19.CondA.Rep1.bam      MCF10A_H3K4me3_chr19.CondB.Rep2.bam      MCF10A_input_chr19.CondA.Rep3.bam
MCF10A_H3K4me3_chr19.CondA.Rep1.bam.bai  MCF10A_H3K4me3_chr19.CondB.Rep2.bam.bai  MCF10A_input_chr19.CondA.Rep3.bam.bai
MCF10A_H3K4me3_chr19.CondA.Rep2.bam      MCF10A_H3K4me3_chr19.CondB.Rep3.bam      MCF10A_input_chr19.CondB.Rep1.bam
MCF10A_H3K4me3_chr19.CondA.Rep2.bam.bai  MCF10A_H3K4me3_chr19.CondB.Rep3.bam.bai  MCF10A_input_chr19.CondB.Rep1.bam.bai
MCF10A_H3K4me3_chr19.CondA.Rep3.bam      MCF10A_input_chr19.CondA.Rep1.bam        MCF10A_input_chr19.CondB.Rep2.bam
MCF10A_H3K4me3_chr19.CondA.Rep3.bam.bai  MCF10A_input_chr19.CondA.Rep1.bam.bai    MCF10A_input_chr19.CondB.Rep2.bam.bai
MCF10A_H3K4me3_chr19.CondB.Rep1.bam      MCF10A_input_chr19.CondA.Rep2.bam        MCF10A_input_chr19.CondB.Rep3.bam
MCF10A_H3K4me3_chr19.CondB.Rep1.bam.bai  MCF10A_input_chr19.CondA.Rep2.bam.bai    MCF10A_input_chr19.CondB.Rep3.bam.bai

CourseData/EPI_data/module123/triplicates/bigWig:
CondA.Rep1.bw  CondA.Rep2.bw  CondA.Rep3.bw  CondB.Rep1.bw  CondB.Rep2.bw  CondB.Rep3.bw

CourseData/EPI_data/module123/triplicates/peaks:
CondA.Rep1_peaks.narrowPeak  CondA.Rep3_peaks.narrowPeak  CondB.Rep2_peaks.narrowPeak
CondA.Rep2_peaks.narrowPeak  CondB.Rep1_peaks.narrowPeak  CondB.Rep3_peaks.narrowPeak

• triplicates were generated from MCF10A_H3K4me3 by choosing a list of exclusive peaks for condA and condB and subsampling replicates accordingly

Lab Completed!

Congratulations! You have completed Lab 1!