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<!DOCTYPE html>
<html>
<head>
<title>Hi-C Data Analysis Bootcamp</title>
<meta charset="utf-8">
<style>
@import url(https://fonts.googleapis.com/css?family=Yanone+Kaffeesatz);
@import url(https://fonts.googleapis.com/css?family=Droid+Serif:400,700,400italic);
@import url(https://fonts.googleapis.com/css?family=Ubuntu+Mono:400,700,400italic);
body { font-family: 'Droid Serif'; }
h1, h2, h3 {
font-family: 'Yanone Kaffeesatz';
font-weight: normal;
}
.column:first-of-type {float:left}
.column:last-of-type {float:right}
.split-40 .column:first-of-type {width: 40%}
.split-40 .column:last-of-type {width: 60%}
.remark-code, .remark-inline-code { font-family: 'Ubuntu Mono'; }
</style>
</head>
<body>
<textarea id="source">
class: center, middle
# Hi-C Data Analysis Bootcamp 2018
## Harvard Medical School, Boston, MA
#### Soo Lee, Nezar Abdennur, Peter Kerpedjiev, Fritz Lekschas
---
# WiFi
* Name: HMS Public
```
Open a browser, type an address (e.g google.com),
then the HMS wifi page will be open instead.
Scroll down to the bottom of the page,
click the button to agree on terms and condition.
```
## Repository
https://github.com/hms-dbmi/hic-data-analysis-bootcamp
## slides
https://hms-dbmi.github.io/hic-data-analysis-bootcamp/#1
---
# Preparation
### Connect to a Linux VM instance on Amazon Cloud
```
ssh <username>@<aws_public_ip>
```
* username: ec2-user
* passwd: bootcamp0508
```bash
ls
```
```bash
anaconda3 certs data hic-data-analysis-bootcamp jupyter_notebooks pandoc-1.19.1 R
```
---
# Docker for Hi-C pipeline and Higlass
* List Docker images on your machine.
```bash
docker images
```
```bash
REPOSITORY TAG IMAGE ID CREATED SIZE
gehlenborglab/higlass v0.2.63 1d9fe99415ac 3 weeks ago 2.7GB
duplexa/4dn-hic v42 a1381e47a736 4 weeks ago 2.6GB
hello-world latest 1815c82652c0 10 months ago 1.84kB
```
---
# Run docker
* Create a Docker container from image `duplexa/4dn-hic:v42`, with a bash entry point
* Mout the `~/data` directory as `/d/` inside the container using the `-v` option
```
ls
ls data
```
```
docker run -it -v ~/data:/d/:rw duplexa/4dn-hic:v42 bash
```
```
ls
ls /d
```
* cd into the data directory
```
cd /d/
```
---
# Practice!
Hi-C data processing pipeline
1. Mapping
* Tools : `bwa`, `samtools`
* output: `bam` file
2. Filtering / sorting / Creating a list of contacts
* Tools: `pairsamtools`, `pairix`
* outputs: `pairs` file
* Intermediate: `pairsam` files
3. Binning & Normalization
* Tools: `cooler`
* outputs: `cool` file
4. Aggregation (for HiGlass)
* Tools: `cooler`
* outputs: `multires.cool` (`mcool`) file
---
# Ingredients
* input Hi-C data in the FASTQ format:
```
input_R1.fastq
input_R2.fastq
```
* bwaIndex (hg38) - reference genome formatted for bwa
```
hg38.fasta.amb
hg38.fasta.ann
hg38.fasta.bwt
hg38.fasta.pac
hg38.fasta.sa
```
* chrom size (hg38) - text file listing the chromosomes and their sizes
```
hg38.mainonly.chrom.size
```
---
# Check input files
```
head data/input_R1.fastq
```
```
@1
GATCACACCACTGCACTCCAGCCTGGGCGACAGAGCAAGACTCTATCTCAAAAAAAAAAAAAAAAATCAAGAAATAATGGCTGAAAATTTCCAAAATTTG
+1
AABBBFFFB@BFGGGFFGGGFGGHHHCGCGGGGHFBGCFFGFFFHHHHHGHGBEE0EEE/EEEEEE03?F33BF4BEB22?2/B2BFD22FFDFGHFCG2
@2
CATCAGAAGGATGGATCCCTTGTGCTACCCATTTTATAGTCACAAACACGTTCCTCACTCCCTGACCCCTGGCAACCACTAATGGGTTTTTCATCTCTGT
+2
CCCCCFFFFFFFGGGGGGGGGGHHHHHHHHHHHHHHHHHHHGHHHHHHHGHHHHHHHGHHHHHHHHHHGGGHHHHHHGGHHEGHHGFFGGGHHHHHHHHE
@3
CTTTTTTTTGTAAAATGAGAAAGCTGGATGAAAGGATCTCTAAGGTCCCTTCACCTTTGAAATCTGATAACTCTAAGGTTTAATTTCCTCATCTGTCAAA
```
```
head data/hg38.mainonly.chrom.size
```
```
chr1 248956422
chr2 242193529
chr3 198295559
chr4 190214555
chr5 181538259
chr6 170805979
chr7 159345973
chr8 145138636
chr9 138394717
chr10 133797422
```
---
# Output files
* they will be generated through this session.
```
output.bam
parsed.pairsam.gz, sorted.pairsam.gz, deduped.pairsam.gz, filtered.pairsam.gz
output.pairs.gz, output.pairs.gz.px2
output.cool
output.mcool
```
* In case you could not generate an output file, but move on to the next step, use our prebaked output files.
```
output.prebaked.bam
parsed.prebaked.pairsam.gz, sorted.prebaked.pairsam.gz
deduped.prebaked.pairsam.gz, filtered.prebaked.pairsam.gz
output.prebaked.pairs.gz, output.prebaked.pairs.gz.px2
output.prebaked.cool
output.prebaked.mcool
```
---
# Alignment!
* Tools :
* `bwa` (http://bio-bwa.sourceforge.net/) : alignment
* `samtools` (https://samtools.github.io/) : format conversion (sam->bam)
* Usage
```bash
bwa mem -SP5M -t8 <index> <fastq1> <fastq2> | samtools view -bhS - > <outbam>
```
* input: `fastq` files, bwa genome index files
* output: `bam` file
* `-SP5M` : Hi-C-specific options
* Add `-t <ncores>` to `bwa mem` to use multiple cores (e.g. `-t 4`)
* Command
```bash
bwa mem -SP5M -t8 hg38.fasta input_R1.fastq input_R2.fastq | \
samtools view -bhS - > output.bam
```
---
# How to read a `bam` file
* A `bam` file is a binary file that stores alignment information for sequenced reads
* Tools :
* `samtools` (https://samtools.github.io/) : viewing a bam file
* Command
```bash
samtools view output.bam | less -S
```
* SAM/BAM format: https://samtools.github.io/hts-specs/SAMv1.pdf
* Example lines
```bash
SRR1658581.203938 81 chr5 76758064 0 51M50S = 76757789 -326 GGGGTTTTACCTTGTTGGCCAGGCTGGTCTTGAACTCCTGACCTCATGATCGATCATCACCATCTTGGTTTTGGTGGGTTTTGGCCGGCTTCCTTACTGCA .BB@DCCC@DCDDBBBDDDDCDDDDDDDDEDCC>DECCEEEFFHGHHHJIGIJJJJHHFIIIJIIJHIJJJJJJJJJJJJJJJIJJJJHHHHHFFFFFCCC NM:i:0 MD:Z:51 AS:i:51 XS:i:48 SA:Z:chr5,76780991,+,51M50S,0,0;
SRR1658581.203938 2113 chr5 76780991 0 51M50H = 76757789 -23203 TGCAGTAAGGAAGCCGGCCAAAACCCACCAAAACCAAGATGGTGATGATCG CCCFFFFFHHHHHJJJJIJJJJJJJJJJJJJJJIHJIIJIIIFHHJJJJIG NM:i:0 MD:Z:51 AS:i:51 XS:i:43 SA:Z:chr5,76758064,-,51M50S,0,0;
SRR1658581.203938 161 chr5 76757789 60 101M = 76758064 326 TTGATTTTTCCTTCTGGACAGGCTCCAATATGGCTCTGTGCAGCACTGTTATTGGTCCTGTCGTTACTGAAAATTCTGATTTTTTTTTTTTGGAAACGGAG CCCFFFFFHHHHHJJJJJJJJJJEHJJJJIIIJFEIJIHIIJIIIGIIHHEIIEGHGHGIFHIHIGIGIIJJHGHHHH;?CEEDDDDDDDD<-4>AC?B85 NM:i:0 MD:Z:101 AS:i:101 XS:i:0
```
---
# Filtering!
* Tools :
* `pairsamtools` (https://github.com/mirnylab/pairsamtools) : filtering
* Steps
* Parse : annotate reads, create a `pairsam` file
* Sort : sort by chromosome1-chromosome2-position1-position2
* Dedup : Mark PCR duplicates
* Select : Filter valid Hi-C reads
* Split : create a `pairs` or `bam` file
* input: `bam` file
* output: `pairs` file
* intermediate: `pairsam` files
---
# Filtering!
* Tools :
* `pairsamtools` (https://github.com/mirnylab/pairsamtools) : filtering
* Pair types
<p style="text-align: center">
<img src="http://pairsamtools.readthedocs.io/en/latest/_images/terminology.png" width="600" />
</p>
---
# Filtering!
* Tools :
* `pairsamtools` (https://github.com/mirnylab/pairsamtools) : filtering
* Pair types
<p style="text-align: center">
<img src="http://pairsamtools.readthedocs.io/en/latest/_images/read_pair_NU_NN.png" width="600" />
</p>
<p style="text-align: center">
<img src="http://pairsamtools.readthedocs.io/en/latest/_images/read_pair_UR.png" width="250" />
</p>
---
# Filtering!
* Tools :
* `pairsamtools` (https://github.com/mirnylab/pairsamtools) : filtering
* Parse / sort
* Usage
```bash
samtools view -h <input_bam> | \
pairsamtools parse -c <chromsize> -o <parsed_pairsam>
pairsamtools sort --nproc 8 -o <sorted_pairsam> <parsed_pairsam>
```
* Commands
```bash
samtools view -h output.bam | \
pairsamtools parse -c hg38.mainonly.chrom.size -o parsed.pairsam.gz
pairsamtools sort --nproc 8 -o sorted.pairsam.gz parsed.pairsam.gz
gunzip -c parsed.pairsam.gz |less -S # check your output file!
```
* input: `bam` file
* output: `pairsam` file
* intermediate: `pairsam` file
---
# Filtering!
* Tools :
* `pairsamtools` (https://github.com/mirnylab/pairsamtools) : filtering
<p style="text-align: center">
<img src="https://s3.amazonaws.com/pkerp/public/img/hic-bootcamp-presentation/pairsam_screen.png" width="700" />
</p>
---
# Filtering!
* Tools :
* `pairsamtools` (https://github.com/mirnylab/pairsamtools) : filtering
* Dedup / Select / Split
* Usage
```bash
pairsamtools dedup --mark-dups -o <deduped_pairsam> <sorted_pairsam>
pairsamtools select <condition> -o <filtered_pairsam> <deduped_pairsam>
pairsamtools split --output-pairs <output_pairs> <filtered_pairsam>
```
* Commands
```bash
pairsamtools dedup --mark-dups -o deduped.pairsam.gz sorted.pairsam.gz
pairsamtools select \
'(pair_type == "UU") or (pair_type == "UR") or (pair_type == "RU")' \
-o filtered.pairsam.gz deduped.pairsam.gz
pairsamtools split --output-pairs output.pairs.gz filtered.pairsam.gz
gunzip -c output.pairs.gz |less -S # check output file!
```
* input: `pairsam` file
* output: `pairs` file
* intermediate: `pairsam` files
---
# Pairs file
* A `pairs` file is a block-gzipped text file that stores list of contact loci
* Tools:
* `pairix` (https://github.com/4dn-dcic/pairix) : indexing & querying `pairs` files
* Commands
```bash
bgzip output.pairs # (if not gzipped)
pairix -f output.pairs.gz # indexing
pairix output.pairs.gz 'chr2:1-60000000|chr4:5000000-6000000' # querying
```
* Pairs format: https://github.com/4dn-dcic/pairix/blob/master/pairs_format_specification.md
* Example lines
```bash
#columns: readID chr1 pos1 chr2 pos2 strand1 strand2
EAS139:136:FC706VJ:2:2104:23462:197393 chr1 10000 chr1 20000 + +
EAS139:136:FC706VJ:2:8762:23765:128766 chr1 50000 chr1 70000 + +
```
---
# Binning! (Generating a matrix)
* Tools:
* `cooler` (https://github.com/mirnylab/cooler)
* Usage
```bash
cooler cload pairix <chrsize_file>:<bin_size> <pairs_file> <out_prefix>.cool
```
* input: `pairs` file, `chromsize` file
* output : `cool` file
* binsize : e.g.) 5000 (high resolution), 500000 (lower resolution)
* Command
```bash
cooler cload pairix hg38.mainonly.chrom.size:500000 output.pairs.gz output.cool
```
---
# How to read a `cool` file
* A `cool` file is an HDF5-format file that stores contact matrices and normailzation vectors.
* Tools:
* `cooler` (https://github.com/mirnylab/cooler)
* Command
```bash
cooler dump -t pixels --header --join -r chr19 output.cool
```
* Cool format : http://cooler.readthedocs.io/en/latest/datamodel.html
---
# Normalization! (Matrix balancing)
* Tools:
* `cooler` (https://github.com/mirnylab/cooler)
* Usage
```bash
cooler balance <cool_file>
```
* Command
```bash
cooler balance output.cool
# check output file! (w/ -b for normalized matrix)
cooler dump -t pixels --header --join -r chr19 -b output.cool
```
* input : `cool` file
* output : `cool` file
---
# Aggregation! (For HiGlass view)
* Tools:
* `cooler` (https://github.com/mirnylab/cooler)
* Usage
```bash
cooler zoomify <cool_file>
```
* Command
```bash
cooler zoomify output.cool
```
* input : `cool` file
* output : `mcool` file
---
# Hi-C pipeline on 4DN Data Portal
* A modular Hi-C mapping pipeline for reproducible data analysis.
* https://data.4dnucleome.org/files-processed/4DNFIC2XS1Y3/
* https://github.com/4dn-dcic/docker-4dn-hic
---
# Visualization
To explore the features of a matrix, it helps to be able to see the data. Example with HiGlass:
<p style="text-align: center">
<img src="img/sox2.png" width="600" />
</p>
---
# Existing tools
<p style="text-align: center">
<table style="margin: 0px auto">
<tr>
<td> Juicebox </td>
<td> <img style="margin-left: 20px" src="img/juicebox-rectangle.png" height="120" /> </td>
</tr>
<tr>
<td>WashU Epigenome Browser</td>
<td><img style="margin-left: 20px" src="img/washu-epigenome-browser.png" height="120" /></td>
</tr>
<tr>
<td> 3D Genome Browser </td>
<td><img style="margin-left: 20px" src="img/3d-genome-browser.png" height="120" /></td>
</tr>
</table>
</p>
---
# HiGlass
<p style="text-align: center">
<img src="img/higlass-overview.png" width=400 />
</p>
HiGlass is a viewer for HiC and other genomic data. It can be used
online (<a href="http://higlass.io/" >http://higlass.io</a>), or run locally with private data.
<h3>
</h3>
---
# HiGlass Architecture
HiGlass uses a client-server achitecture to allow users to view large contact
matrices in a web browser.
<p style="text-align: center">
<img src="img/higlass-architecture.png" width=400 />
</p>
---
# Capabilities
HiGlass is designed to facilitate the comparison of multiple genomic datasets through
"tracks" and "views".
<p style="text-align: center">
<img src="img/g12878_vs_K257.png" width=600/>
</p>
---
# Anatomy of HiGlass
<table>
<tr>
<td>
<p style="text-align: center">
<img src="img/higlass-anatomy-11.png" width=400/>
</p>
</td>
<td>
<p style="margin-left: 15px">
HiGlass is composed of a collection of views and
tracks.
</p>
</td>
</tr>
</table>
---
# Anatomy of HiGlass
<table>
<tr>
<td>
<p style="text-align: center">
<img src="img/higlass-anatomy-10.png" width=400/>
</p>
</td>
<td>
<p style="margin-left: 15px">
Each view is composed of a set of tracks which share
common axes.
</p>
</td>
</tr>
</table>
---
# Anatomy of HiGlass
<table>
<tr>
<td>
<p style="text-align: center">
<img src="img/higlass-anatomy-9.png" width=400/>
</p>
</td>
<td>
<p style="margin-left: 15px">
Settings related to the view can be accessed via the
"view settings" menu.
</p>
</td>
</tr>
</table>
---
# Anatomy of HiGlass
<table>
<tr>
<td>
<p style="text-align: center">
<img src="img/higlass-anatomy-8.png" width=400/>
</p>
</td>
<td>
<p style="margin-left: 15px">
New views can be created by cloning an existing view.
</p>
</td>
</tr>
</table>
---
# Anatomy of HiGlass
<table>
<tr>
<td>
<p style="text-align: center">
<img src="img/higlass-anatomy-7.png" width=400/>
</p>
</td>
<td>
<p style="margin-left: 15px">
Views can be closed by clicking on the "close view" button ("X").
</p>
</td>
</tr>
</table>
---
# Anatomy of HiGlass
<table>
<tr>
<td>
<p style="text-align: center">
<img src="img/higlass-anatomy-6.png" width=400/>
</p>
</td>
<td>
<p style="margin-left: 15px">
The contents of a view can be modified by adding tracks.
</p>
</td>
</tr>
</table>
---
# Anatomy of HiGlass
<table>
<tr>
<td>
<p style="text-align: center">
<img src="img/higlass-anatomy-5.png" width=400/>
</p>
</td>
<td>
<p style="margin-left: 15px">
Tracks can be layed out horizontally where they track
the horizontal axis.
</p>
</td>
</tr>
</table>
---
# Anatomy of HiGlass
<table>
<tr>
<td>
<p style="text-align: center">
<img src="img/higlass-anatomy-4.png" width=400/>
</p>
</td>
<td>
<p style="margin-left: 15px">
Tracks can also be layed out vertically where they
track the vertical axis.
</p>
</td>
</tr>
</table>
---
# Anatomy of HiGlass
<table>
<tr>
<td>
<p style="text-align: center">
<img src="img/higlass-anatomy-3.png" width=400/>
</p>
</td>
<td>
<p style="margin-left: 15px">
And tracks can be placed in the center, where they move
along both axes.
</p>
</td>
</tr>
</table>
---
# Anatomy of HiGlass
<table>
<tr>
<td>
<p style="text-align: center">
<img src="img/higlass-anatomy-2.png" width=400/>
</p>
</td>
<td>
<p style="margin-left: 15px">
Track settings can be adjusted via the track settings
menu.
</p>
</td>
</tr>
</table>
---
# Anatomy of HiGlass
<table>
<tr>
<td>
<p style="text-align: center">
<img src="img/higlass-anatomy-1.png" width=400/>
</p>
</td>
<td>
<p style="margin-left: 15px">
Overlays can be added on top of existing tracks by clicking
on the '+' icon of the track toolbar.
</p>
</td>
</tr>
</table>
---
# Anatomy of HiGlass
<table>
<tr>
<td>
<p style="text-align: center">
<img src="img/higlass-anatomy-0.png" width=400/>
</p>
</td>
<td>
<p style="margin-left: 15px">
Tracks can be closed by clicking on the 'X' of the
track toolbar.
</p>
</td>
</tr>
</table>
---
# Practical exercise
Re-create this figure:
<p style="text-align: center">
<img src="https://cloud.githubusercontent.com/assets/2143629/24535688/a6043f1a-15a3-11e7-8197-d5d3ce227bc6.png" height=400/>
</p>
---
# Creating a local instance
Start a HiGlass instance and expose it to the outside world using the `--site-url` parameter.
```
higlass-manage start --site-url $PUBLIC_IP
```
---
# Creating a local instance
Start a HiGlass instance and expose it to the outside world using the `--site-url` parameter.
```
higlass-manage start --site-url $PUBLIC_IP
```
## Add data
```
higlass-manage ingest ~/data/Schwarzer-et-al-2017-UNTR.multi.cool
higlass-manage ingest ~/data/Schwarzer-et-al-2017-NIPBL.multi.cool
higlass-manage ingest ~/data/Schwarzer-et-al-2017-RNAseq-minus.bw
```
---
class: center, middle
## Navigate to HiGlass website
Get the IP of the AWS instance hosting HiGlass:
```
[ec2-user@ip-172-31-26-229 ~]$ echo $PUBLIC_IP
54.145.184.38
```
And navigate to it using port 8989:
<p style="text-align: center">
<video width="500" loop="loop" autoplay="autoplay">
<source src="https://s3.amazonaws.com/pkerp/public/img/hic-bootcamp-presentation/navigate-to-higlass-url.mp4" type="video/mp4">
</video>
</p>
---
# Toggle the position bar
The genome position search bar serves to show the visible extent of the data.
It can also be used to navigate to a particular locus.
<p style="text-align: center">
<video width="800" loop="loop" autoplay="autoplay">
<source src="https://s3.amazonaws.com/pkerp/public/img/hic-bootcamp-presentation/toggle-position-search-box.mp4" type="video/mp4">
</video>
</p>
* Note that just because an assembly is selected, that doesn't constrain the
data shown. It is up to the user to add datasets from a matching assembly.
---
# Add a 1D track and change its type
* To start, we'll add the RNA-Seq dataset as a track on top of the view:
* After adding it, we'll change its type to `horizontal-bar` (from `horizontal-line`)
<p style="text-align: center">
<video width="500" loop="loop" autoplay="autoplay">
<source src="https://s3.amazonaws.com/pkerp/public/img/hic-bootcamp-presentation/add-top-bar-track.mp4" type="video/mp4">
</video>
</p>
---
# Add a heatmap
Next we'll add one of the heatmaps to the center of the view:
<p style="text-align: center">
<video width="500" loop="loop" autoplay="autoplay">
<source src="https://s3.amazonaws.com/pkerp/public/img/hic-bootcamp-presentation/add-heatmap-track.mp4" type="video/mp4">
</video>
</p>
---
# Add gene annotations
We'll add gene annotations to the top.
<p style="text-align: center">
<video width="500" loop="loop" autoplay="autoplay">
<source src="https://s3.amazonaws.com/pkerp/public/img/hic-bootcamp-presentation/add-gene-annotations.mp4" type="video/mp4">
</video>
</p>
---
# Resize the current view and replicate it
* We want to create four views in total showing two different datasets
* To start, we'll resize the current view and replicate it twice
<p style="text-align: center">
<video width="500" loop="loop" autoplay="autoplay">
<source src="https://s3.amazonaws.com/pkerp/public/img/hic-bootcamp-presentation/resize-and-clone-twice.mp4" type="video/mp4">
</video>
</p>
---
# Replace existing dataset and copy it
* Now we'll replace one of the datasets and clone that view as well
* Since we already have the NIPBL condition, we'll replace the second view with UNTR (wt)
<p style="text-align: center">
<video width="500" loop="loop" autoplay="autoplay">
<source src="https://s3.amazonaws.com/pkerp/public/img/hic-bootcamp-presentation/resize-and-clone-twice.mp4" type="video/mp4">
</video>
</p>
---
# Lock zoom and location
* Let's make sure the top two rows and the bottom two rows always show the same locations as each other
<p style="text-align: center">
<video width="500" loop="loop" autoplay="autoplay">
<source src="https://s3.amazonaws.com/pkerp/public/img/hic-bootcamp-presentation/take-and-lock-zoom-and-location.mp4" type="video/mp4">
</video>
</p>
---
# Go to interesting locations
## Zoom top to:
By providing a set of genomic coordinates, we can zoom to a defined region
in the heatmap.
```
chr14:50,143,364-88,897,098 & chr14:70,582,768-97,882,196 [offset 0,0:0,0]
```
## Zoom bottom to:
```
chr14:66,384,132-76,643,845 & chr14:67,964,160-79,229,728 [offset 0,0:0,0]
```