Short Read Workshop Day 1 - Experimental Design Example 1: How to log in to vieques

Syllabus Day 1: Exp. Design Instructors: Mary Allen/Tim Read Day 2: Intro Linux & Editors Instructors: David Knox/Joey Azofeifa Day 3: QC, barcodes, intro mapping Instructors: David Brazel /Amber Sorenson Day 4: Moving, clusters, notes Instructors: Joey Azofeifa /Daniel Malmer Day 5: Mapping and viz Instructors: Jess Vera /Phil Richmond Day 6: Reseq Instructors: Phil Richmond /Aaron Odell Day 7: Txpn (RNA-seq) Instructors: Aaron Odell/Mary Allen Day 8: Peak calling (ChIP) Instructors: Li Wang /Amber Sorenson Week 1Week 2

Course structure Before class: – Videos ( – Firefox does not always work. Use Chrome, Internet Explorer or Safari During class (9 am-12 pm) – Examples with example files (fastq, sam, bam, bed) After class (12-5 pm) – Homework

You will be learning “command line” This is not a GUI Spelling and Capitalization matter! Google can help you learn “command line unix” – The names for commands are not always intuitive (you will learn many this week)

Login Mac or unix Open Terminal by clicking on the magnifying glass in the corner and typing terminal and hitting enter ssh –X – Type password – When/if it says “enter passphrase” just hit enter 2x!!! PC – open Putty – Type vieques.colorado.edu under the host name – Hit open – Type identikey – Type password – When/if it says “enter passphrase” just hit enter 2x!!!

Check your access pwd ls mkdir mine touch temp.txt ls ls -lahtr cd mine pwd ls -lahtr cd.. pwd

VPN (Virtual Private Network) If you are not on campus to connect to vieques you must have vpn installed and turn it on before you log in! – Otherwise the login will just hang. internet-services/vpn/

Why use a server (cluster) Many computers can often do work much faster than one computer can. – built to run on a server/cluster (multi-threading) Some things take lots of space or memory Most bioinformatic programs are written for Unix/Linux – Because this platform is by programmers for programmers Often installing is a pain!

Which server can I use? Biofrontiers – Vieques CU Boulder – RC (Resource computing) Pro: they deal with up keep/installing and its mostly free Con: Under maintenance a lot, built more for physics problems Off campus – We don’t know… (Ask department?, Cloud computing?)

Big verse small Class will use small files for the sake of time. If you are using human or mouse data you will likely have much larger files.

In Class problem 1 You want to whole sequence the human genome to find a dominant heterozygous mutation. How deep do you need to sequencing it (1x, 4x, 40x?) How many reads do you need? How many lanes of seq do you need assuming you will get 200 million reads per lane? Hint: human genome is 3 billion bp How much disk space will you use? Where will you process the data?

How to determine coverage Coverage = (number of reads * read length) Size of the genome

In Class problem 2 You are trying to sequence non- coding RNAs that are 100 bp long from penguins. Should you use paired end or single end? Should you use 50 bp or 100 bp? Which RT would you use? Which RT primer would you use?

In Class Problem 3 You would like to ChIP-seq the transcription factor p53.

Your project What experiment have you done or are you planning to do? What method are you using? Which sequencer? How many lanes will you sequence? Will you barcode? Inline or in adapter? Will you do Single vs. paired end? How many replicates will you do? What controls are you using? How much disk space will you use? Where will you process the data?