modify a genbank file for mapping

Insert between the following tags at the tail of your Genbank file the nucleotide FASTA sequence only:

ORIGIN

(sequence goes here)

//

CLC mapping

At ths point, the most current version of the CLC command-line utils is located here:

/usr/local/packages/clc-ngs-cell/

with the mapper for 454 / Sanger reads being clc_ref_assemble_long.

As usual, you need to copy the license.properties file from that folder to your working directory to perform any sort of analysis.

Here are some of the pitfalls:

- making a soft link to the mapping executable will throw a missing license error. forget the soft link, you need to use the fully qualified path of the executable.

- mapping to a Genbank file didn't work. However mapping to a FASTA file completed with blazing quickness.

Once the mapping has producea CAS output, you need to process it to get it in table format:

/usr/local/packages/clc-ngs-cell/assembly_table -n -s input.cas > output.table

SVN stuff

This is how you create a repository (usually done by IT):

svnadmin create (repo_name)

This is how you add content to a repository:

svn import (files | folder) (repo_name) -m "initial import"

This is how you checkout the latest version of something existing in SVN into the current working directory:

svn checkout (repo_name/path)

This is how you check something into it to update the svn with a more current version:

from within the root folder when doing svn checkout (ie: /workspace/genomeQC):

svn commit -m "update to ver 1.5"

FASTQ tools

Here are my favorite FASTQ processing utils:

FASTQC:

To run a QC on your data.

http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/

To run in batch mode with zipped output:

java* -Xmx250m -cp <> uk.ac.bbsrc.babraham.FastQC.FastQCApplication <>

*: make sure that java is at least version 1.6

FASTX Toolkit:

Bunch of scripts for processing the fastq files and qc, but fastqc is better for qc'ing.

http://hannonlab.cshl.edu/fastx_toolkit/download.html

Illumina Technote: How Illumina Sequencing works

Here's a a PDF that describes how Illumina seqeuncing works with nice graphics:
http://www.illumina.com/documents/products/datasheets/datasheet_genomic_sequence.pdf

A copy is saved here:

http://sites.google.com/site/webhostingoffiles/Illumina_datasheet_genomic_sequence.pdf

Another great article detailing sequencing technology is saved here:

http://sites.google.com/site/webhostingoffiles/Sequencingtechnologies%E2%80%94NextGen_nrg2626.pdf

FASTQC

This java app reads a fastq file and generated various quality metrics.

It can be run from both the command-line or a GUI. It is also pretty fast - processing over 4M reads in under 8 seconds - but I ran it on a beast of a machine with 256 GB of RAM.

It is available for download from this site:

http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/

Here is the command-line:
/usr/local/bin/java-1.6.0 -Xmx250m -cp /usr/local/devel/BCIS/external_software/FastQC uk.ac.bbsrc.babraham.FastQC.FastQCApplication < input.fastq >

Where the text in blue represents the installation location.

The output will be a set of HTML and image files zipped inside a folder named < input >_fastqc.

Sun grid engine submission

Here's the typical command I use for making grid submissions:

SGE submission:

qsub -P -m a -M $USER\@jcvi.org -l < fast|medium|default queue > -V -cwd -shell n -b y < command >

Using CLC Bio's command-line tools

This is what I've learned:

In order to run CLC Bio programs from the command line, you need to have a license.properties file in your current working directory. But not any license.properties file, the one associated to the specific version of software you are using. For example, if you are using a command that is on the path, you need to track down which license.properties file it is associated with.

Mapping reads - (greater than 50 bp in length):

For mapping reads back to a reference which have 90% identity over 90% of the length of the query

/usr/local/packages/clc-ngs-cell-2.0.5-linux_64/clc_ref_assemble_long -o < output.cas > -q < query.fasta > -d < database.fasta > -l 0.9 -s 0.9

The output of this mapping, or reference-based assembly, is a .cas file. Getting a table of hits, a la nucmer, you need to process the .cas file with another CLC tool: assembly_table

Processing .cas files:

/usr/local/packages/clc-ngs-cell-2.0.5-linux_64/assembly_table -n -s < output.cas > > < table.tsv >

Dear Diary,

This blog is mostly for me, to have an easy way to find the often used command lines and pipelines, with all their nifty little parameters, that I have to use on a daily basis.

But if this blog offers any insight or suggestions to resolve a problem you're having, then by all means, buy me a beer.