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Revision as of 14:25, 13 November 2014
, 14:25, 13 November 2014no edit summary
'''Note:''' the latest version of this practical is available at: [[SeqShop: Aligning Your Own Genome]]
* The ones here is the original one from the June workshop (updated to be run from elsewhere)
== First Things First ==
== First Things First ==
*Helpful reference to many tools:
*Helpful reference to many tools:
* Practice setting up and running GotCloud on your own.
* Practice setting up and running GotCloud on your own.
== Step 1 : Looking at your FASTQs ==
{{SeqShopLogin}}
== I didn't get sequenced, what can I do? ==
I prepared some test files for you.
* I took a 1000g sample and reduced it to 2x.
** I already created an align.2x.index file for you.
*** 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.
To be consistent with everyone else you can do:
mkdir ~/personal
cp -r /home/mktrost/seqshop/inputs/2x/* ~/personal/.
ls ~/personal/
ls ~/personal/fastq
== I got sequence, how to I get my data ready to run? ==
=== Finding your FASTQs ===
Your FASTQ files are under your <code>personal</code> directory.
Your FASTQ files are under your <code>personal</code> directory.
[[File:FastqlistAnnotated.png]]
[[File:FastqlistAnnotated.png]]
=== Checking your index file listing your FASTQs ===
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
On success it prints: <code>Congratulations, your fastq index looks valid</code>
== Generating the index file listing your FASTQs ==
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.
=== Generating the index file listing your FASTQs ===
What columns do we need in our file that tells GotCloud about our FASTQ?
What columns do we need in our file that tells GotCloud about our FASTQ?
* MERGE_NAME
* MERGE_NAME
We will store our FASTQ info file in: ~/personal/align.2x.index.
We will store our FASTQ info file in: ~/personal/align.2x.index.
=== Using a Spreadsheet ===
There are a few ways to create this file.
* Write into a text file one fastq pair at a time.
* Copy fastq1s into a spreadsheet, fill it in and copy back to a text file
* [[#Using a Script|Write/Use a script]]
==== Using a Regular Text File ====
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).
* Remember to put a tab between each field.
==== Using a Spreadsheet ====
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.
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.
* Thanks to those who thought to do this yesterday - it was a great idea.
* Thanks to those who thought to do this yesterday - it was a great idea.
First, open Excel
First, open Excel
==== Header Row ====
===== Header Row =====
Create the header line by typing each of the column names in a row (you may be able to copy this line):
Create the header line by typing each of the column names in a row (you may be able to copy this line):
* make sure you enter these in all CAPS & spelling does matter
* make sure you enter these in all CAPS & spelling does matter
[[File:HdrRow.png]]
[[File:HdrRow.png]]
==== MERGE_NAME ====
===== MERGE_NAME =====
MERGE_NAME is just your sample name
MERGE_NAME is just your sample name
* Type your Sample name under the MERGE_NAME column, for example: <code>Sample_12345</code>
* Type your Sample name under the MERGE_NAME column, for example: <code>Sample_12345</code>
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.
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.
==== FASTQ1 ====
===== FASTQ1 =====
FASTQ1 is just the 1st in pair FASTQs (or the single FASTQ in single end)
FASTQ1 is just the 1st in pair FASTQs (or the single FASTQ in single end)
* Our sequencing core indicated 1st in pair by <code>R1</code> in the filename.
* Our sequencing core indicated 1st in pair by <code>R1</code> in the filename.
[[File:HdrSheetMN1.png|700]]
[[File:HdrSheetMN1.png|700]]
====FASTQ2====
=====FASTQ2=====
As mentioned before, FASTQ2 files are the 2nd in pair.
As mentioned before, FASTQ2 files are the 2nd in pair.
* They have the same filename as FASTQ1, except replace the R1 with R2
* They have the same filename as FASTQ1, except replace the R1 with R2
[[File:HdrSheetFQ2 2.png]]
[[File:HdrSheetFQ2 2.png]]
==== RGID ====
===== RGID =====
We want to group our FASTQs by Run & Lane.
We want to group our FASTQs by Run & Lane.
* Each Run/Lane combination should have a unique Read Group
* Each Run/Lane combination should have a unique Read Group
[[File:HdrSheetRGannotated.png]]
[[File:HdrSheetRGannotated.png]]
====SAMPLE ====
=====SAMPLE =====
Put your sample name in each row of this column (you can copy from MERGE_NAME)
Put your sample name in each row of this column (you can copy from MERGE_NAME)
==== LIBRARY ====
===== LIBRARY =====
Put your sample name in each row of this column (you can copy from MERGE_NAME)
Put your sample name in each row of this column (you can copy from MERGE_NAME)
* If a sample has multiple library preparations done on it, you would want to give unique names
* If a sample has multiple library preparations done on it, you would want to give unique names
** That is not our case, so just put in the sample name.
** That is not our case, so just put in the sample name.
==== PLATFORM ====
===== PLATFORM =====
Your data was sequenced on ILLUMINA, so enter <code>ILLUMINA</code> in each row of the platform column.
Your data was sequenced on ILLUMINA, so enter <code>ILLUMINA</code> in each row of the platform column.
[[File:HdrSheetDone.png]]
[[File:HdrSheetDone.png]]
==== Copy to Text File ====
===== Copy to Text File =====
Open nedit or your favorite linux editor
Open nedit or your favorite linux editor
nedit ~/personal/align.2x.index&
nedit ~/personal/align.2x.index&
You now have a tab delimited align.2x.index file (a little simpler than yesterday).
You now have a tab delimited align.2x.index file (a little simpler than yesterday).
==== Using a Script ====
When generating an index of your FASTQs, it can be easiest to have a script.
* Especially if you have many samples/runs, it would be very tedious to do by hand
If you are good at scripting, this may be even easier than doing it by hand
* If you aren't good at scripting, and you have too much data to do by hand
** Make friends with someone who is :-)
** I always find it useful to start from another script (reminds me of commands/tricks)
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:
perl /home/mktrost/seqshop/inputs/buildIndex.pl ~/personal > ~/personal/align.2x.index
* <code>></code> means to direct the output to the file specified after the <code>></code>
Curious what the script looks like and what it does in case you want to create one in the future?
<div class="mw-collapsible mw-collapsed" style="width:200px">
<li>View Annotated Script</li>
<div class="mw-collapsible-content">
[[File:BuildIndex.png|800px]]
</div>
</div>
=== Checking your index file listing your FASTQs ===
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
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.
== Create your GotCloud Configuration File ==
== Create your GotCloud Configuration File ==
* Everything else is configured already.
* Everything else is configured already.
[[File:Gc2xconf.png]]
[[File:Gc2xconf.png]]
You'll notice that this file is very similar to the one we have been using.
You'll notice that this file is very similar to the one we have been using.
:* Type Ctrl-a Esc and you should be able to scroll up with your mouse wheel
:* Type Ctrl-a Esc and you should be able to scroll up with your mouse wheel
:** 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)
:** 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)
== Log Out ==
If you have not detached from screen:
Ctrl-a d
exit PuTTY
== FEEDBACK! ==
Since I didn't send this out yesterday, today's survey has feedback for Tuesday & Wednesday.
https://docs.google.com/forms/d/1qaLHq9w1Ib3FZq0CtlrbK_-breNiqGRV06oRYNmUuME/viewform
