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SeqShop: Aligning Your Own Genome, June 2014 (view source)

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'''Note:''' the latest version of this practical is available at: [[SeqShop: Aligning Your Own Genome]]
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* The ones here is the original one from the June workshop (updated to be run from elsewhere)
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== First Things First ==
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*Helpful reference to many tools:
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** http://infoplatter.wordpress.com/2014/04/06/bioinformaticians-pocket-reference/
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*** links to "cheat-sheets", including, Unix, screen, and vi
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* Our wiki with some brief description of how to do some basic commands
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**http://genome.sph.umich.edu/wiki/Basic_Linux_Intro
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* [[Screen Commands]] for commands to use screen (one way to leave your commands running even after you log out)
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== Goals of This Session ==
 
== Goals of This Session ==
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Learn how to go from your FASTQ files to generate Aligned BAMs.
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* Practice setting up and running GotCloud on your own.
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{{SeqShopLogin}}
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== I didn't get sequenced, what can I do? ==
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I prepared some test files for you.
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* I took a 1000g sample and reduced it to 2x.
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** I already created an align.2x.index file for you.
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*** This is a 1000g sample, so the filenames/RG information for these do not match the ones produced by our sequencer that are described below.
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To be consistent with everyone else you can do:
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mkdir ~/personal
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cp -r /home/mktrost/seqshop/inputs/2x/* ~/personal/.
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ls ~/personal/
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ls ~/personal/fastq
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== I got sequence, how to I get my data ready to run? ==
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=== Finding your FASTQs ===
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Your FASTQ files are under your <code>personal</code> directory.
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Look at your directory:
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ls ~/personal
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* <code>ls</code> does a directory listing
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* <code>~/</code> means to start from your home/base directory
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** Using ~/ means the command will work even if you have changed directories
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You should see 2 directories: Run_992 & Run_993
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* Our DNA was run through as 2 sequencing runs.
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What is under those directories?  Let's check:
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ls ~/personal/Run_*
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* <code>*</code> is a Linux wild-card match
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** It will do a directory listing on both Run directories at the same time:
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[[File:RunDir.png]]
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You will see your sample name instead of <code>12345</code>.
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Since we found another directory, let's check below that for our fastqs:
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ls ~/personal/Run_*/*
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You should see your fastq files:
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[[File:Fastqlist.png]]
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Those filenames are cryptic, what do they mean?
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[[File:FastqlistAnnotated.png]]
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=== Checking your index file listing your FASTQs ===
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Are you analyzing your own genome?  Do you think you setup your file correctly?
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Try running this script to see if you have any errors:
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perl /home/mktrost/seqshop/inputs/checkIndex.pl ~/personal/align.2x.index
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On success it prints: <code>Congratulations, your fastq index looks valid</code>
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NOTE: This script is tailored to the filenames provided by our sequencing core as described above.
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* It could be tailored to other methods, but is designed for the paths of our data.
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=== Generating the index file listing your FASTQs ===
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What columns do we need in our file that tells GotCloud about our FASTQ?
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* MERGE_NAME
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* FASTQ1
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* FASTQ2
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* RGID
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* SAMPLE
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* LIBRARY
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* PLATFORM
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We will store our FASTQ info file in: ~/personal/align.2x.index.
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There are a few ways to create this file.
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* Write into a text file one fastq pair at a time.
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* Copy fastq1s into a spreadsheet, fill it in and copy back to a text file
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* [[#Using a Script|Write/Use a script]]
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==== Using a Regular Text File ====
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Follow the instructions below, but do it one FASTQ1 at a time (you won't be able to paste a full column of FASTQs at a time).
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* Remember to put a tab between each field.
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==== Using a Spreadsheet ====
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Since we just have a handful of FASTQs, we can use a spreadsheet to construct our file and then copy the data into a text file.
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* Thanks to those who thought to do this yesterday - it was a great idea.
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First, open Excel
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===== Header Row =====
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Create the header line by typing each of the column names in a row (you may be able to copy this line):
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* make sure you enter these in all CAPS & spelling does matter
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MERGE_NAME FASTQ1 FASTQ2 RGID SAMPLE LIBRARY PLATFORM
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[[File:HdrRow.png]]
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===== MERGE_NAME =====
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MERGE_NAME is just your sample name
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* Type your Sample name under the MERGE_NAME column, for example: <code>Sample_12345</code>
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[[File:FastqHdrMN.png]]
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All FASTQs are for the same sample, so you will use <code>Sample_12345</code> on every line.  We will fill those in after we get know how many rows we need.
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===== FASTQ1 =====
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FASTQ1 is just the 1st in pair FASTQs (or the single FASTQ in single end)
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* Our sequencing core indicated 1st in pair by <code>R1</code> in the filename.
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** All our FASTQs are paired end (no single end fastqs)
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* To get a a list of just the <code>R1</code> files:
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ls -1 ~/personal/Run_*/*/*R1*
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* The -1 option tells <code>ls</code> to list the matching files in a single column
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** If you have noticed, the number of columns in a directory listing varies based on the width of your window.
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** We want to copy these files into one column of a spreadsheet so want them displayed as a single column
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Highlight this list and copy them into the FASTQ1 column of your spreadsheet:
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[[File:HighlightedFASTQs.png]]
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[[File:HdrSheetFQ1.png|700]]
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Now that we know how many rows we have, copy your sample name into all rows:
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[[File:HdrSheetMN1.png|700]]
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=====FASTQ2=====
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As mentioned before, FASTQ2 files are the 2nd in pair.
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* They have the same filename as FASTQ1, except replace the R1 with R2
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You could do an ls and copy & paste as we did for FASTQ1, BUT we'd have to make sure we properly matched up the mates.
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EASIER solution:
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* In your spreadsheet, copy your FASTQ1 filenames into the FASTQ2 column:
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[[File:HdrSheetFQ2 1.png]]
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* Now, replace <code>R1</code> with <code>R2</code> in JUST the FASTQ2 column
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** Make sure FASTQ1 column keeps the <code>R1</code>
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[[File:HdrSheetFQ2 2.png]]
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===== RGID =====
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We want to group our FASTQs by Run & Lane.
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* Each Run/Lane combination should have a unique Read Group
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** Run is in the directory path: Run_992 or Run_993
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** Lane is indicated by L001/L002
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*** Lanes may have multiple FASTQ pairs
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**** the sequencing core split them into smaller FASTQs, but they are the same Run/Lane, so should have the same read group.
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Populate the RGID column with a name unique to each Read Group/Lane combination:
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[[File:HdrSheetRGannotated.png]]
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=====SAMPLE =====
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Put your sample name in each row of this column (you can copy from MERGE_NAME)
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===== LIBRARY =====
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Put your sample name in each row of this column (you can copy from MERGE_NAME)
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* If a sample has multiple library preparations done on it, you would want to give unique names
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** That is not our case, so just put in the sample name.
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===== PLATFORM =====
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Your data was sequenced on ILLUMINA, so enter <code>ILLUMINA</code> in each row of the platform column.
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[[File:HdrSheetDone.png]]
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===== Copy to Text File =====
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Open nedit or your favorite linux editor
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nedit ~/personal/align.2x.index&
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Click <code>New File</code> in the pop-up stating that it couldn't find that file.
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Copy (Ctrl-c) your table from EXCEL (including the header row, but with no extra rows/columns.
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Paste (Ctrl-v) into your nedit window.
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Navigate through the file - the columns should be delimited with tabs.
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Save (Ctrl-s) & close nedit.
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You now have a tab delimited align.2x.index file (a little simpler than yesterday).
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==== Using a Script ====
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When generating an index of your FASTQs, it can be easiest to have a script.
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* Especially if you have many samples/runs, it would be very tedious to do by hand
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If you are good at scripting, this may be even easier than doing it by hand
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* If you aren't good at scripting, and you have too much data to do by hand
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** Make friends with someone who is :-)
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** I always find it useful to start from another script (reminds me of commands/tricks)
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If you still need to create your file and you don't want to use the spreadsheet method above, you can run a script that I made:
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perl /home/mktrost/seqshop/inputs/buildIndex.pl ~/personal > ~/personal/align.2x.index
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* <code>></code> means to direct the output to the file specified after the <code>></code>
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Curious what the script looks like and what it does in case you want to create one in the future?
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<div class="mw-collapsible mw-collapsed" style="width:200px">
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<li>View Annotated Script</li>
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<div class="mw-collapsible-content">
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[[File:BuildIndex.png|800px]]
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</div>
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</div>
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=== Checking your index file listing your FASTQs ===
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Are you analyzing your own genome?  Do you think you setup your file correctly?
 +
 +
Try running this script to see if you have any errors:
 +
perl /home/mktrost/seqshop/inputs/checkIndex.pl ~/personal/align.2x.index
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 +
On success it prints: <code>Congratulations, your fastq index looks valid</code>
 +
 +
NOTE: This script is tailored to the filenames provided by our sequencing core as described above.
 +
* It could be tailored to other methods, but is designed for the paths of our data.
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== Create your GotCloud Configuration File ==
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Now that you have your FASTQ info file created, we need to setup the configuration.
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'''If you updated your gotcloud.2x.conf file yesterday, you still need to do this step'''
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* We updated a few settings to match 1000g
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*# Updated the version of the genome reference
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*# Updated to use BWA instead of BWA_MEM
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*# Updated the version of BWA
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You can copy from my directory to your personal directory:
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cp /home/mktrost/seqshop/inputs/gotcloud.2x.conf ~/personal/.
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You need to update the conf file to find your align.2x.index and ensure your output directory is setup.
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nedit ~/personal/gotcloud.2x.conf &
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(or use your favorite editor)
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* Change the <code>IN_DIR</code> line, replacing <code>YOUR_USER_NAME</code> with your user name.
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* Everything else is configured already.
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[[File:Gc2xconf.png]]
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You'll notice that this file is very similar to the one we have been using.
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* Just a few modifications to run a new test on the whole genome
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== Run your Alignment ==
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=== Screen ===
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The alignment pipeline will run overnight, but you'll want to log out.
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; How do I leave something running on the server even if I log out?
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: One solution is screen!
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; How do I use screen?
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: Before running your command, you need to start screen:
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: <pre>screen</pre>
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[[File:Screen.png]]
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As it says, press <code>Space</code> or <code>Return</code>.
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* It should now look basically the same as your normal command line.
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You can now start your alignment:
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/home/mktrost/seqshop/gotcloud/gotcloud align --conf ~/personal/gotcloud.2x.conf --numjobs 1
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Yes, leave that as /home/mktrost/seqshop/gotcloud/gotcloud - that is where gotcloud is installed.  The ~/... points GotCloud to your specific configuration file.
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You should now see your alignment running:
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;Want to log out and leave your job running?
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In the screen window, type:
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Ctrl-a d
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(Hold down Ctrl and type 'a', let go of both and type 'd')
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* This will "detach" from your screen session while your alignment continues to run.
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;How do you log back into screen tomorrow?
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screen -r
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This will resume an already running screen.
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* Feel free to test it out and you will see your alignment still running
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** Just use Ctrl-a d to detach from screen and leave your job running
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; Scrolling problems?
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: If you want to scroll and screen doesn't scroll like you normally would?
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:* Type Ctrl-a Esc and you should be able to scroll up with your mouse wheel
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:** Or at least that is what I do from my Linux machine - (sorry I'm typing this up/testing these commands from Linux and not windows, so can't test it out)
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== Log Out ==
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If you have not detached from screen:
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Ctrl-a d
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exit PuTTY
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== FEEDBACK! ==
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Since I didn't send this out yesterday, today's survey has feedback for Tuesday & Wednesday.
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https://docs.google.com/forms/d/1qaLHq9w1Ib3FZq0CtlrbK_-breNiqGRV06oRYNmUuME/viewform
Retrieved from "http://genome.sph.umich.edu/wiki/Special:MobileDiff/10870...11970"

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