1. Facilitator: Richard Bruskiewich
NGS Bioinformatics Workshop 1.2 Tutorial – Sequence Formats, Databases and Visualization Tools
March 15th, 2012
BioSci room B9242
Facilitator: Richard Bruskiewich
Adjunct Professor, MBB

2. Learning Objectives Linux revisited
Quick dive into the Open-Bio pool (BioPython)
A first look at NGS data:
NCBI short read archive
Processing NGS: FASTX tool kit et al.
Visualization: IGV

3. Files and Permission Linux user permissions: owner, group, or others
Owner/user is the person who created the file
“OWNS” the file / directory
Group is a team of people that’s associated together
GROUP project / Team work
Others is just other people on the server
Each file / directory can have it’s permission set to (r)ead, (w)rite, or e(x)ecute

4. chmod: change file permissions
Do a long listing (ls –l)
dr-x-wxrw- Separated into four sections
(d)(r - x)(- w x)(r w -)
Examples: chmod o+x foo.txt  grant ‘execute’ permission to ‘others’ on foo.txt
chmod g-rw foo.txt  remove ‘read’ and ‘write’ permission from group
chmod ugo+rwx foo.txt  grant all rights to everyone
To change the user/group (‘owner’) of a file:
chmod ubuntu:ubuntu foo.txt
directory or file (-)
user (owner)
group
others

5. a few useful tips…
Hitting “tab” will auto-complete file or program names (or suggest possible names)
Up arrow will let you return to previous commands
Editing of text files: “nano” is an easier alternative to “emacs”, but less powerful  alternatively, use SSH client to transfer files on your Windows desktop, edit them in Windows, then transfer back  BUT: make sure you use a text editor that knows the difference between a Windows and a Linux text file (e.g. Notepad++)

6. Some more useful basic Linux commands
“cd” changes your directory, e.g. ‘cd /usr/local’
“man” display manual for command, e.g. ‘man ‘ls’
“pwd” tells you the directory you are currently in (= working directory)
“history” will list recent commands, enumerated with line numbers. By; typing an exclamation point with the line number (e.g. !123), you can redo the command

7. Accessing remote servers
“ssh” – Secure Shell
ssh –i private_keypair
“scp” – Secure CoPy
ssh –i private_keypair
Where user is the account (default: local user)
and host is the internet name of the computer (defaults: local host)

8. OpenBio Case Study: BioPython

9. NGS Bioinformatics Workshop 1
NGS Bioinformatics Workshop 1.2 Tutorial – Sequence Formats, Databases and Visualization Tools
First look at ngs data

11.
Linux, MacOSX or Unix only

12. Get the precompiled binary
wget
fastx_toolkit_0.0.13_binaries_Linux_2.6_amd64.tar.bz2
bunzip2 fastx_toolkit_0.0.13_binaries_Linux_2.6_amd64.tar.bz2
tar –xvf fastx_toolkit_0.0.13_binaries_Linux_2.6_amd64.tar
sudo mv bin/* /usr/local/bin

13. FASTX tool kit I FASTQ-to-FASTA converter FASTQ Information
Convert FASTQ files to FASTA files.
FASTQ Information
Chart Quality Statistics and Nucleotide Distribution
FASTQ/A Collapser
Collapsing identical sequences in a FASTQ/A file into a single sequence (while maintaining reads counts)
FASTQ/A Trimmer
Shortening reads in a FASTQ or FASTQ files (removing barcodes or noise).
FASTQ/A Renamer
Renames the sequence identifiers in FASTQ/A file.
FASTQ/A Clipper
Removing sequencing adapters / linkers

14. FASTX tool kit II FASTQ/A Reverse-Complement FASTQ/A Barcode splitter
Producing the Reverse-complement of each sequence in a FASTQ/FASTA file.
FASTQ/A Barcode splitter
Splitting a FASTQ/FASTA files containing multiple samples
FASTA Formatter
Changes the width of sequences line in a FASTA file
FASTA Nucleotide Changer
Converts FASTA sequences from/to RNA/DNA
FASTQ Quality Filter
Filters sequences based on quality
FASTQ Quality Trimmer
Trims (cuts) sequences based on quality
FASTQ Masker
Masks nucleotides with 'N' (or other character) based on quality

16. Integrative Genomics Viewer