A Beginners Guide to Call Somatic Mutation

class: center, middle, inverse, title-slide

# A Beginners Guide to Call Somatic Mutation
## Part I
### Lijia Yu
### 2020/10/02 (updated: 2020-10-03)

---

# Outline

1. Introduction to linux for bioinformatics
2. A brief Introduction of Cancer Genomics
3. Quality Control

---

1. open broswer link: 192.168.0.105:8787

![](./2019-06-17-Introduction_to_Linux_for_Bioinformatics_files/figure/RStudio.png)

---

# UNIX file system

![](./2019-06-17-Introduction_to_Linux_for_Bioinformatics_files/figure/UnixFileSystem.png)

---
# UNIX file system

## ls

`ls` is a command to list computer files in Unix and Unix-like operating systems.

```bash
ls /home/admin
```

![](./2019-06-17-Introduction_to_Linux_for_Bioinformatics_files/figure/ls.png)

---

# UNIX file system: files and extensions

- For some operating systems, file extensions are important and define the file type. In e.g. Windows files have a three/four letter extension(e.g. .jpg, .exe, .docx, …)

- In UNIX file extensions are arbitrary (no particular sense), at least for the operating system. A file can have several extensions and will be recognized by the file permissions and content.

- Popular file extensions in bioinformatics:
  - scripts
     - .sh (shell/bash) 
     - .pl (perl) 
     - .py (python) 
     - .r (R)
  - data
     - .txt (text)
     - .csv/.tsv (tabular)
     - .fasta/.fastq (sequence files)

.footnote[
[1] [Linux for bioinformatics
](https://data.bits.vib.be/pub/trainingen/Linux/Linux_01_2018.pdf)
]

---

# UNIX file system: users and access rights

Every file and directory is protected. A set of permissions determines who can access a certain file and what kind of access is allowed.

- User: the user who owns the file

- Group: other users from the same group

- Others: all others in the system

- Read: display the file

- Write: display and modify the content of the file

- Execute: run a file ( only for scripts and compiled programs)

.footnote[
[1] [Linux for bioinformatics
](https://data.bits.vib.be/pub/trainingen/Linux/Linux_01_2018.pdf)
]

---

# UNIX file system: users and groups

```bash
ls -a
```
print a list of all files and directories (incl. hidden files). Hidden files can be recognized by
‘.’ at the start of the name, ‘..’ is the parent directory

```
[root@nccl /home/lirui]$ ls -a
.  ..  .bash_logout  .bash_profile  .bashrc
```

![](./2019-06-17-Introduction_to_Linux_for_Bioinformatics_files/figure/ls-a.png)

.footnote[
[1] [Linux for bioinformatics
](https://data.bits.vib.be/pub/trainingen/Linux/Linux_01_2018.pdf)
]

---

# UNIX file system: users and groups

```bash
ls -l /usr/bin/gpg
```

```
lrwxrwxrwx 1 root root 4 Feb 18 11:03 /usr/bin/gpg -> gpg2
```

- file permissions,
- number of hard links,
- owner name,
- owner group,
- file size,
- time of last modification, and file/directory name

.footnote[
[1] [What do the fields in ls -al output mean?
](https://unix.stackexchange.com/questions/103114/what-do-the-fields-in-ls-al-output-mean)
]

---

# UNIX file system: users and groups

File permissions is displayed as following;

![](./2019-06-17-Introduction_to_Linux_for_Bioinformatics_files/figure/access_right.png)

- r = readable
- w = writable
- x = executable

.footnote[
[1] [What do the fields in ls -al output mean?
](https://unix.stackexchange.com/questions/103114/what-do-the-fields-in-ls-al-output-mean)

[2] [Linux for bioinformatics
](https://data.bits.vib.be/pub/trainingen/Linux/Linux_01_2018.pdf)
]

---

# UNIX file system: file permission

An example -rwxrw-r--, this means the line displayed is:

- a regular file (displayed as -)
--

- readable, writable and executable by owner (rwx)
--

- readable, writable, but not executable by group (rw-)
--

- readable but not writable or executable by other (r--)

.footnote[
[1] [Linux for bioinformatics
](https://data.bits.vib.be/pub/trainingen/Linux/Linux_01_2018.pdf)
]

---

# UNIX file system

When entering a command you can use a wildcard character, this is used a substitute for one or many other characters. They are often used with file and directory name and filesystem commands.

- `*` Match any number of characters
- `?` Match one character
- `[]` specify a range of characters on that position
- `{}` specify a list of terms - separated by commas
- `!` Exclude this range of characters

.footnote[
[1] [Linux for bioinformatics
](https://data.bits.vib.be/pub/trainingen/Linux/Linux_01_2018.pdf)
]

---

# UNIX file system

How many files will you find using the following command:

- `ls -l *.c`
- `ls -l bam*`
- `ls -l samtools.?`
- `ls -l *.*[!a]`
- `ls -l {*.c,*.h}`
- `ls -l version.[a-z]h`

.footnote[
[1] [Linux for bioinformatics
](https://data.bits.vib.be/pub/trainingen/Linux/Linux_01_2018.pdf)
]

---

# Cancer genomics

- `Cancer genomics` is the study of the totality of DNA sequence and gene expression differences between tumour cells and normal host cells.

- `Cancer` is a group of genetic diseases that result from changes in the genome of cells in the body, leading them to grow uncontrollably.

.center[<img src="https://www.genome.gov/sites/default/files/inline-images/dd_03_full.jpg" height="200">]

.footnote[
[1] [Cancer Genomics](https://www.genome.gov/dna-day/15-ways/cancer-genomics)]

---

# Germline versus Somatic Mutations

.center[<img src="https://ib.bioninja.com.au/_Media/somatic-vs-germline_med.jpeg" height="400">]

.footnote[
[1] [Germline versus Somatic Mutations](https://ib.bioninja.com.au/standard-level/topic-3-genetics/33-meiosis/somatic-vs-germline-mutatio.html)]

---

# Tests to Detect Cancer

- **biopsies** or **operations** where tiny parts of the cancer tissue are removed for study.
- **Liquid biopsies**: detection of circulating tumor DNA (or ctDNA) in the **blood of patients**

# Sequencing platform

- [Illumina](https://www.illumina.com/) (HiSeq/MiSeq/NextSeq/NovaSeq, etc.)
- [Thermo Fisher](https://www.thermofisher.com/hk/en/home.html) (Ion Torrent)
- [BGI](https://en.mgitech.cn/) (MGISEQ)
- [PacBio](https://www.pacb.com/) (Sequel)

---

# Illumina sequencing

.center[<img src="https://hackteria.org/wiki/images/thumb/1/19/Chemistry.png/569px-Chemistry.png" height="400">]

.footnote[
[1] [HiSeq2000 - Next Level Hacking](https://hackteria.org/wiki/HiSeq2000_-_Next_Level_Hacking)]

---

# FASTA format

In bioinformatics and biochemistry, the FASTA format is a text-based format for representing either nucleotide sequences or amino acid (protein) sequences, in which nucleotides or amino acids are represented using single-letter codes.

```bash
>SEQUENCE_1
MTEITAAMVKELRESTGAGMMDCKNALSETNGDFDKAVQLLREKGLGKAAKKADRLAAEG
LVSVKVSDDFTIAAMRPSYLSYEDLDMTFVENEYKALVAELEKENEERRRLKDPNKPEHK
IPQFASRKQLSDAILKEAEEKIKEELKAQGKPEKIWDNIIPGKMNSFIADNSQLDSKLTL
>P01013 GENE X PROTEIN (OVALBUMIN-RELATED-fakedata)
QIKDLLVSSSTDLDTTLVLVNAIYFKGMWKTAFNAEDTREMPFHVTKQESKPVQMMCMNNSFNVATL
KMKILELPFASGDLSMLVLLPDEVSDLERIEKTINFEKLTEWTNPNTMEKRRVKVYLPQMKIEEKYN
VLMALGMTDLFIPSANLTGISSAESLKISQAVHGAFMELSEDGIEMAGSTGVIEDIKHSPESEQFRA
FLFLIKHNPTNTIVYFGRYWSP
>DNA_SEQ1
NNATTGCCGDTTGTACCtgcgtgtcagac
```

.footnote[
[1] [FASTA format used in blast](https://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastDocs&DOC_TYPE=BlastHelp)
]

---

# Flowcell

.center[<img src="2020-10-02-A_beginners_guide_to_Call_somatics_mutation_Part_I_files/figure/Flowcell.png" height="400">]

.footnote[
[1] [Flowcell imaging](https://is.muni.cz/el/1431/podzim2018/Bi5444/um/Bi5444_Analysis_of_sequencing_data_lesson_03.pdf)]

---

# FASTQ format

A FASTQ file normally uses four lines per sequence.

- Line 1 begins with a '@' character and is followed by a sequence identifier and an optional description (like a FASTA title line).
- Line 2 is the raw sequence letters. A,T,C,G,N.
- Line 3 begins with a '+' character and is optionally followed by the same sequence identifier (and any description) again.
- Line 4 encodes the quality values for the sequence in Line 2, and must contain the same number of symbols as letters in the sequence.

A FASTQ file containing a single sequence might look like this:

```bash
@SEQ_ID
GATTTGGGGTTCAAAGCAGTATCGATCAAATAGTAAATCCATTTGTTCAACTCACAGTTT
+
!''*((((***+))%%%++)(%%%%).1***-+*''))**55CCF>>>>>>CCCCCCC65
```

---

# FASTQ format

## Illumina sequence identifiers

**@HWUSI-EAS100R:6:73:941:1973#0/1**

| Name | Meaning|
|---------------|---------------------------------------------------------------------|
| HWUSI-EAS100R | the unique instrument name                                          |
| 6             | flowcell lane                                                       |
| 73            | tile number within the flowcell lane                                |
| 941           | 'x'-coordinate of the cluster within the tile                       |
| 1973          | 'y'-coordinate of the cluster within the tile                       |
| #0            | index number for a multiplexed sample (0 for no indexing)           |
| /1            | the member of a pair, /1 or /2 (paired-end or mate-pair reads only) |

---

# Quality

A quality value `Q` is an integer mapping of `P` (i.e., the probability that the corresponding base call is incorrect). Two different equations have been in use. The first is the standard Sanger variant to assess reliability of a base call, otherwise known as Phred quality score:

`$$Q_{sanger}=-10log_{10}P$$`

`$$Q_{solexa}=-10log_{10}\frac{P}{1-P}$$`

---

# Phred Quality Score

| Phred Quality Score | Probability of incorrect base call | Base call accuracy |
|---------------------|------------------------------------|--------------------|
| 10                  | 1 in 10                            | 90%                |
| 20                  | 1 in 100                           | 99%                |
| 30                  | 1 in 1000                          | 99.9%              |
| 40                  | 1 in 10,000                        | 99.99%             |
| 50                  | 1 in 100,000                       | 99.999%            |
| 60                  | 1 in 1,000,000                     | 99.9999%           |

---

# Encoding

![](./2019-06-17-Introduction_to_Linux_for_Bioinformatics_files/figure/Phred.png)

---

# FASTQ

```
@NCCL-000000f0-ba51-4443-9096-3fd0e846a363/1
GATATGGGTGAATATAGAAAAGTCTTCAAATAAAAAAATA
+
cccccghhhghhhhhhhhhhhhhhhhhhhhhhhhhhhhhh
@NCCL-00000acb-689a-4a96-8a0c-2b4de1c1e294/1
CATATTTTGGTTATGTTGTAAAATTTCTTCACTCTGAATG
+
caac_eebec[_g^b[egghhghhgPeggehhfghhfdhd
@NCCL-00000aed-7f8d-46b6-be65-ef860566b017/1
GGTGGTAGAATGTCCAGATGAAAGCTTCATTCAACCCATC
+
_a[c`gP[e_dgghgdebfgdbeb^_f_ffdg^bPbdgge
@NCCL-00000bf3-b23f-4649-811f-6a58cc1507cd/1
GTGTCCTTCCCTTCTTCCTATCCTCTGCCACTGCCTCGCT
+
ccccchhhhhhhhhhhhhhhhghhhhghhhhh^ghgghhh
```

---

# Quick Quitz

1. How many reads in `/opt/data/Illumina_normal_R1.fq.gz` file?

Could you give me an answer using one line command?

(5 mins)

---

# Quick Quitz

1. How many reads in `/opt/data/Illumina_normal_R1.fq.gz` file?

- [How to count fastq reads](https://www.biostars.org/p/139006/)

```bash
#yourfile.fastq  
echo $(cat yourfile.fastq|wc -l)/4|bc

#yourfile.fastq.gz
echo $(zcat yourfile.fastq.gz|wc -l)/4|bc
```

---

# Command line: echo

## Display line of text/string that are passed as an argument .

```bash
echo -e "Geeks \bfor \bGeeks"
```

.footnote[
[1] [echo command in Linux with Examples](https://www.geeksforgeeks.org/echo-command-in-linux-with-examples/)
]

---

# Command line: cat and zcat

```bash
cat /home/admin/database/reference/human/hg19/chr1.fa
zcat /opt/data/Illumina_normal_R1.fq.gz
```
Writing context to standard output.

`zcat` is a command line utility for viewing the contents of a compressed file without literally uncompressing it.

---

# Command line: bc

## command line calculator

`bc` command is used for command line calculator. It is similar to basic calculator by using which we can do basic mathematical calculations.

```bash
echo "12+5" | bc
echo "10^2" | bc
```

.footnote[
[1] [bc command in Linux with examples](https://www.geeksforgeeks.org/bc-command-linux-examples/)
]

---

# Single end vs Paired end

.center[<img src="https://www.yourgenome.org/sites/default/files/images/illustrations/bioinformatics_single-end_pair-end_reads_yourgenome.png" width="350">]
.footnote[
[1] [Question: paired end illumina reads](https://www.biostars.org/p/162806/)
]

---

# Quality Control

.center[<img src="https://blog.omictools.com/wp-content/uploads/2017/11/NGS-QC-figure-omictools.png" width="550">]

.footnote[
[1] [Best bioinformatics software for RNA-seq quality control](https://omictools.com/blog/your-top-3-rna-seq-quality-control-tools)
]
---

# Quality Control

## tools

**1.FASTQC** (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)

A quality control tool for high throughput sequence data.

**2.FASTX-Toolkit** (http://hannonlab.cshl.edu/fastx_toolkit/)

The FASTX-Toolkit is a collection of command line tools for Short-Reads FASTA/FASTQ files preprocessing.

**3.FASTP** (https://github.com/OpenGene/fastp)

An ultra-fast all-in-one FASTQ preprocessor (QC/adapters/trimming/filtering/splitting/merging...)

---

#  Command line: wget

## Download files from website/ftp

- `wget` is the non-interactive network downloader

```bash
wget -c https://www.bioinformatics.babraham.ac.uk/projects/fastqc/fastqc_v0.11.9.zip

```

.footnote[
[1] [Wget command in Linux/Unix](https://www.geeksforgeeks.org/wget-command-in-linux-unix/)
]

# Command line: unzip and gunzip

## Unzip file

```bash
unzip fastqc_v0.11.9.zip
```

---

# Install source code

- 
```bash
cd package-directory
```
- find README or Install related document, then **read it**!
- 
```bash
chmod 755 fastqc
```

---

# Create directory and symbolic link

```bash
cd ~
mkdir -p ./project/out
mkdir -p ./project/tmp
mkdir -p ./project/src
mkdir -p ./project/in

cd ./project/in

ln -rs /opt/data/*.gz ./
```

The `ln` command is a standard Unix command utility used to create a hard link or a symbolic link to an existing file.

- `-s, --symbolic` make symbolic links instead of hard links
- `-r, --relative` create symbolic links relative to link location

---

# File Path

### Absolute Path

```bash
/home/admin/software/FastQC/fastqc -t 1 \
-o /home/yulijia/project/out \
--noextract \
-d /home/yulijia/project/tmp \
-f fastq \
/home/admin/ILLUMINA_FASTQ/cancer_R1.fq.gz \
/home/admin/ILLUMINA_FASTQ/cancer_R2.fq.gz 
```

An absolute path is defined as the specifying the location of a file or directory from the root directory(`/`). 1
To write an absolute path-name:
- Start at the root directory (`/`) and work down.
- Write a slash (`/`) after every directory name (last one is optional)

.footnote[
[1] [Absolute and Relative Pathnames in UNIX](https://www.geeksforgeeks.org/absolute-relative-pathnames-unix/)
]
---

# File Path

### Relative Path

```bash
/home/admin/software/FastQC/fastqc -t 1 \
-o ../out \
--noextract \
-d ../tmp \
-f fastq \
../in/cancer_R1.fq.gz \
../in/cancer_R2.fq.gz 
```

Relative path is defined as the path related to the present working directly(`pwd`). It starts at your current directory and never starts with a `/` .

---

# FASTQC

- `-t --threads`

Specifies the number of files which can be processed
simultaneously.  Each thread will be allocated 250MB of
memory so you shouldn't run more threads than your
available memory will cope with, and not more than
6 threads on a 32 bit machine

---

# FASTQC

- `-o --outdir`

Create all output files in the specified output directory.
Please note that this directory must exist as the program
will not create it.  If this option is not set then the 
output file for each sequence file is created in the same
directory as the sequence file which was processed.

---

# FASTQC

- `--noextract`

Do not uncompress the output file after creating it.  You
should set this option if you do not wish to uncompress
the output when running in non-interactive mode.

---

# FASTQC

- `-d --dir`

Selects a directory to be used for temporary files written when
generating report images. Defaults to system temp directory if
not specified.

---

# FASTQC

- `-f --format`

Bypasses the normal sequence file format detection and forces the program to use the specified format. Valid formats are bam, sam, bam_mapped, sam_mapped and fastq.

---

# FASTQC

```bash
SYNOPSIS

fastqc [-o output dir] [--(no)extract] [-f fastq|bam|sam] 
       [-c contaminant file] seqfile1 .. seqfileN
```

---

# Quiz

1. Run FastQC for all the data in `/opt/data` directory.

2. Try to download fastp and install it in your `/home/yourname` directory.

---

.footnote[
* All pictures without footnote are getting from Wikipedia or browser snapshots.
]