Evaluating and processing raw sequencing data GVA2017
Overview
Before you start the alignment and analysis processes, it can be useful to perform some initial quality checks on your raw data. If you don't do this (or even if you do), you may notice later that something looks fishy in the the output: for example, many of your reads are not mapping or the ends of many of your reads do not align. Both can give you clues about whether you need to process the reads to improve the quality of data that you are putting into your analysis.
For many years this tutorial is discussed at some length for if it should be included as a main tutorial, if it should be included as an optional tutorial, or if it should be ignored all together as the quality of data increases. Last year a colleague of ours spent several days working with and trying to understand some data he got back before reaching out for help, after a few hours of running into a wall, fastqc was used to determine that the library was not constructed correctly in less than 30 minutes. Thus cementing the information as an important tutorial for a quick check which may save you significant amounts of time later on.
Learning Objectives
This tutorial covers the commands necessary to use several common programs for evaluating read files in FASTQ format and for processing them (if necessary).
- Introduction to the development nodes (and idev sessions) on TACC.
- Diagnose common issues in FASTQ read files that will negatively impact analysis.
- Trim adaptor sequences and low quality regions from the ends of reads to improve analysis.
Table of Contents
Interactive development (idev) sessions
As we discussed in our first tutorial the head node is a space shared by all and we don't like stepping on each others toes. While the launcher_creator.py helper script makes working with the compute nodes much easier, they still take time to initiate a run (waiting in the que) and if you have errors in your commands your job will fail and you will lose your place in line. An idev (or interactive development session) is a way to move off the head node and onto a single compute node, but work interactively to see if your commands actually work, give you much quicker feedback, and if everything goes as you hope, your data. idev sessions are much more limited in duration and in general its not necessary to see every line a program spits out once you are familiar with the type of data you will get. Additionally, we are going to use a priority access reservation set up special for the BDIB summer school that you normally would not have access to but should guarantee immediate starting of your idev session.
Copy and paste the following command, and read through the commented lines to make sure it is function correctly:
idev -m 240 -r CCBB_5.22.17PM -A UT-2015-05-18 # This should return the following: # We found an ACTIVE reservation request for you, named CCBB_5.22.17PM. # Do you want to use it for your interactive session? # Enter y/n [default y]: # If for any reason you don't see the above message let us know by raising your hand. # Your answer should be y, which should return the following: # Reservation : --reservation=CCBB_5.22.17PM (ACTIVE) # Some of you may see a new prompt stating something like the following: # We need a project to charge for interactive use. # We will be using a dummy job submission to determine your project(s). # We will store your (selected) project $HOME/.idevrc file. # Please select the NUMBER of the project you want to charge.\n # 1 OTHER_PROJECTS # 2 UT-2015-05-18 # Please type the NUMBER(default=1) and hit return: # If you see this message, again let us know. # You will then see something similar to the following: # job status: PD # job