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| Creosote genome sequencing and assembly notes:
| | ==Assembly== |
| | | [[Creosote Assembly]] |
| *Sample obtained from front yard of 4951 W. McElroy Dr.
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| *Sequences obtained from one lane of Illumina HiSeq2000
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| *Fastq files delivered from UAGC
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| **82 files
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| **Headers are Sanger format (code 33)
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| ***Description of Fastq file format with notes on specific decoding of header names generated by various technologies: http://en.wikipedia.org/wiki/FASTQ_format
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| **Pairend reads
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| ***lane3_NoIndex_L003_R1_041.fastq
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| ***lane3_NoIndex_L003_R2_041.fastq
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| **Need to get adapter sequences used in sequencing
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| ***TGACCA (Not present in sequence reads)
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| *Check quality with fastqc: http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/
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| **[[Creosote First Run FastQC]]
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| '''Steps:'''
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| *Merge R1 files; merge R2 files
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| *gzip them
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| '''Trim sequences'''
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| *Get this package of Haibaos:
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| ** git clone git://github.com/tanghaibao/jcvi.git
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| ** SET PATH: export PYTHONPATH=/lib/python (which is the dir above jcvi)
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| ** may need to install biopython: sudo easy_install biopython
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| *Run this: python -m jcvi.apps.baseclean trim R1.fastq.gz R2.fastq.gz
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| **Automatically trims and cleans sequences, also does the conversion to appropriate fastq format
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| **NOTE: This program should download trimmomatic, but may need to update the path of the timmomatic program in the program
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| *If the Trimmer script fails for silly reasons, you can run it from the command-line:
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| java -Xmx4g -cp Trimmomatic-0.13/trimmomatic-0.13.jar org.usadellab.trimmomatic.TrimmomaticPE lane3_NoIndex_L003_R1_001.b64.fastq.gz lane3_NoIndex_L003_R2_001.b64.fastq.gz lane3_NoIndex_L003_R1_001.pairs.fastq.gz lane3_NoIndex_L003_R1_001.frags.fastq.gz lane3_NoIndex_L003_R2_001.pairs.fastq.gz lane3_NoIndex_L003_R2_001.frags.fastq.gz ILLUMINACLIP:adapters.fasta:2:40:15 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
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| '''Genome Assembly'''
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| *Note: Bao recommends CLC for genome assembly. Runs faster, less memory, less sensitive to bad data. Compute intensive. THIS IS COMMERCIAL SOFTWARE
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| '''Running SOAPdenovo'''
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| SOAPdenovo31mer all -s ../../soap.config.eric -o SoapAssem -K 25 -p 16 -R -d -D -F
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| **Note: if SOAP crashes, try another XXmer binary (e.g. 63mer)
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| '''Running Velvet'''
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| **Need to interleave reads:
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| ~/src/velvet_1.1.04/shuffleSequences_fastq.pl lane3_NoIndex_L003_R1_001.pairs.fastq lane3_NoIndex_L003_R2_001.pairs.fastq merged_pairs.fastq
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| **set threading of velvet with env var
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| export OMP_NUM_THREADS=32
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| **running velveth
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| OMP_NUM_THREADS=32 velveth VelvetAssem 31 -shortPaired -fastq.gz merged_pairs.fastq.gz -short -fastq.gz lane3_NoIndex_L003_R1_001.frags.fastq.gz -short -fastq.gz lane3_NoIndex_L003_R2_001.frags.fastq.gz
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| OMP_NUM_THREADS=32 velvetg VelvetAssem -scaffolding yes -exp_cov auto -cov_cutoff auto -min_contig_lgth 200 -ins_length 150
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| '''Other Stuff'''
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| '''Trimming reads'''
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| *Trim Paired ends with Trimmomatic: http://www.usadellab.org/cms/index.php?page=trimmomatic
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| *Assumes Illumina Encoding (code: 64, not code: 33)
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| **Need to convert for the HighSeq Reads:
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| ** easy_install biopython
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| ** git clone git://github.com/tanghaibao/jcvi.git
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| ** export PYTHONPATH=/lib/python (which is the dir above jcvi)
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| ** python -m jcvi.formats.fastq (Install missing packages)
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| '''Cleaning Single Reads:'''
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| *Sequences cleaned using trimReads by Haibao Tang: https://github.com/tanghaibao/trimReads/tree/
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| **Ran with supplied adapter sequence file:
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| >Adapter 4
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| TGACCA
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| >Adapter 4 rc
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| TGGTCA
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| **Command-line run:
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| Running /home/elyons/bin/trimReads -Q 33 -f /home/elyons/projects/genome/data/creosote/Sample_lane3/adapter/adapter.faa ./lane3_NoIndex_L003_R2_015.fastq
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| '''Converting sequences'''
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| *python -m jcvi.formats.fastq convert (read help file, default conversion Sanger (code 33) to Illumina (code 64)
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| '''Other programs to clean sequences'''
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| *python -m jcvi.apps.baseclean trim fastqfile (single ended)
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| *python -m jcvi.apps.baseclean trim R1.fastq.gz R2.fastq.gz (paired ended)
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| '''keep sequences in single files (or two files for a pair of reads)'''
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| *Cat all the R1s together
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| *Cat all the R2s together
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