Illumina GA: Difference between revisions
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cat s_8_?_????_qseq.txt | /usr/local/bin/qseq2fastq.pl > /media/SeqStore2/AL006/s_8_sequence.txt & | cat s_8_?_????_qseq.txt | /usr/local/bin/qseq2fastq.pl > /media/SeqStore2/AL006/s_8_sequence.txt & | ||
**In many cases, we need to repeat the base calling using the GA Pipeline because some of the lanes either contain libraries that have biased base composition (bisulfite converted libraries) or the libraries were overloaded. In such cases, we need to us one of the "good lane" to determine the phasing for the base caller. This requires running | **In many cases, we need to repeat the base calling using the GA Pipeline because some of the lanes either contain libraries that have biased base composition (bisulfite converted libraries) or the libraries were overloaded. In such cases, we need to us one of the "good lane" to determine the phasing for the base caller. This requires running Bustard and Gerald in the genome-miner. Note that in the second command line you have to set the control lane to the best lane of the flowcell. | ||
cd /media/NewReads/FreshReads/091209_AL006 | cd /media/NewReads/FreshReads/091209_AL006 | ||
/usr/local/GAPipeline-1.5.1/bin/bustard.py Data/Intensities/ --CIF --control-lane=4 --GERALD=../config.txt -make | /usr/local/GAPipeline-1.5.1/bin/bustard.py Data/Intensities/ --CIF --control-lane=4 --GERALD=../config.txt -make |
Revision as of 23:58, 27 December 2009
Illumina sequencing
Sample information
- Each sample must have the following information:
- Sample ID: for sample tracking, please adopt a consistent naming scheme. A name should have the following fields separated by hyphens "-". Each field should not contain any hyphen. Use underscore "_" as a separator if necessary. The sample ID is often too long to write on the tube. It's okay to use a shorter name on the tube, but there has to be a look-up table on the wiki page to map a short name to the Sample ID. The full-length IDs have to be used in the GA tracking form.
- Initial of the person who prepared the sample.
- Methods used for the sample processing, such as Exome, BisSeq, MDA etc.
- Names of the starting materials, such as CV_iPS, PGP1 etc.
- Batch ID or date of the library preparation.
- Additional information.
Here is an example: KZ-Exome-NA12878-Oct16-EL140.3
- For external libraries, please use the following fields:
- Institution name;
- The PI's initial;
- Label on the tube;
- Date of receipt.
- PAGE gel image with Invitrogen LowMass ladder as the reference.
- Record of QPCR quantification.
Workflow for post-run processing
- The data for each run is stored in the folder /media/NewReads/FreshReads of genome-miner.ucsd.edu
- The run root folder name looks like: 091209_HL029 or 091218_AL006.
- The SCS software reports sequences in the qseq format by default.
- Before you run the following commands, you need the sysadmin privilege:
sudo bash
- The .qseq files can be found in the [Run Root Folder]/Data/Intensities/BaseCalls folder, such as
/media/NewReads/FreshReads/091209_HL029/Data/Intensities/BaseCalls
- Most downstream processing requires the sequences in the fastq format, one file per lane. There are two options to obtain fastq files:
- Fistly, create a new folder in the SeqStore directory:
mkdir /media/SeqStore2/AL006
- If you trust the basecaller of the SCS software, then you may simply use a format converter to generate fastq files:
cd /media/NewReads/FreshReads/091209_HL029/Data/Intensities/BaseCalls cat s_1_?_????_qseq.txt | /usr/local/bin/qseq2fastq.pl > /media/SeqStore2/AL006/s_1_sequence.txt & cat s_2_?_????_qseq.txt | /usr/local/bin/qseq2fastq.pl > /media/SeqStore2/AL006/s_2_sequence.txt & . . . cat s_8_?_????_qseq.txt | /usr/local/bin/qseq2fastq.pl > /media/SeqStore2/AL006/s_8_sequence.txt &
- In many cases, we need to repeat the base calling using the GA Pipeline because some of the lanes either contain libraries that have biased base composition (bisulfite converted libraries) or the libraries were overloaded. In such cases, we need to us one of the "good lane" to determine the phasing for the base caller. This requires running Bustard and Gerald in the genome-miner. Note that in the second command line you have to set the control lane to the best lane of the flowcell.
cd /media/NewReads/FreshReads/091209_AL006 /usr/local/GAPipeline-1.5.1/bin/bustard.py Data/Intensities/ --CIF --control-lane=4 --GERALD=../config.txt -make cd Data/Intensities/Bustard1.5.1_21-12-2009_root #Note that the name of Bustard directory contains the date the previous command was run. nohup make recursive -j 8 & # this will take a few hours # when the base calling is completed, you may find the eight fastq files in the following folders: cd /media/NewReads/FreshReads/091218_AL006/Data/Intensities/Bustard1.5.1_21-12-2009_root/GERALD_21-12-2009_root cp s_?_sequence.txt > /media/SeqStore2/AL006 # A summary of base calling is in the file Summary.htm.