Difference between revisions of "Creosote Assembly"
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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 | 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 | ||
OMP_NUM_THREADS=32 velvetg VelvetAssem -scaffolding yes -exp_cov auto -cov_cutoff auto -min_contig_lgth 200 -ins_length 150 | OMP_NUM_THREADS=32 velvetg VelvetAssem -scaffolding yes -exp_cov auto -cov_cutoff auto -min_contig_lgth 200 -ins_length 150 | ||
| + | #FULL RUN | ||
| + | OMP_NUM_THREADS=32 velveth VelvetAssem 31 -shortPaired -fastq.gz merged_pairs.fastq.gz -short -fastq.gz R1_all.frags.fastq.gz -short -fastq.gz R2_all.frags.fastq.gz | ||
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Revision as of 10:53, 20 August 2011
Creosote genome sequencing and assembly notes:
- Sample obtained from front yard of 4951 W. McElroy Dr.
- Sequences obtained from one lane of Illumina HiSeq2000
- Fastq files delivered from UAGC
- 82 files
- Headers are Sanger format (code 33)
- Description of Fastq file format with notes on specific decoding of header names generated by various technologies: http://en.wikipedia.org/wiki/FASTQ_format
- Pairend reads
- lane3_NoIndex_L003_R1_041.fastq
- lane3_NoIndex_L003_R2_041.fastq
- Need to get adapter sequences used in sequencing
- TGACCA (Not present in sequence reads)
- Check quality with fastqc: http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/
Steps:
- Merge R1 files; merge R2 files
- gzip them
Convert sequence formats (I haven't tried this yet)
Haibao Tang 8/5/11 11:09 AM on the conversion, i found an alternative: zcat R1_all.fastq.gz | seqret fastq-sanger::stdin fastq-illumina::stdout | gzip > R1_converted.fastq.gz install emboss just tested, takes 20-30 minutes on R1_all
Trim sequences
- Get this package of Haibaos:
- git clone git://github.com/tanghaibao/jcvi.git
- SET PATH: export PYTHONPATH=/lib/python (which is the dir above jcvi)
- may need to install biopython: sudo easy_install biopython
- Run this: python -m jcvi.apps.baseclean trim R1.fastq.gz R2.fastq.gz
- Automatically trims and cleans sequences, also does the conversion to appropriate fastq format
- NOTE: This program should download trimmomatic, but may need to update the path of the timmomatic program in the program
- If the Trimmer script fails for silly reasons, you can run it from the command-line:
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
Genome Assembly
- Note: Bao recommends CLC for genome assembly. Runs faster, less memory, less sensitive to bad data. Compute intensive. THIS IS COMMERCIAL SOFTWARE
Running SOAPdenovo
SOAPdenovo31mer all -s ../../soap.config.eric -o SoapAssem -K 25 -p 16 -R -d -D -F
- Note: if SOAP crashes, try another XXmer binary (e.g. 63mer)
Running Velvet
- Need to interleave reads:
#test run ~/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 #full data with modified shuffleSequences script that handles gzip files ~/src/velvet_1.1.04/shuffleSequences_fastq_gzip.pl R1_all.pairs.fastq.gz R2_all.pairs.fastq.gz merged_pairs.fastq.gz
- set threading of velvet with env var
export OMP_NUM_THREADS=32
- running velveth
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 OMP_NUM_THREADS=32 velvetg VelvetAssem -scaffolding yes -exp_cov auto -cov_cutoff auto -min_contig_lgth 200 -ins_length 150 #FULL RUN OMP_NUM_THREADS=32 velveth VelvetAssem 31 -shortPaired -fastq.gz merged_pairs.fastq.gz -short -fastq.gz R1_all.frags.fastq.gz -short -fastq.gz R2_all.frags.fastq.gz
Other Stuff
Trimming reads
- Trim Paired ends with Trimmomatic: http://www.usadellab.org/cms/index.php?page=trimmomatic
- Assumes Illumina Encoding (code: 64, not code: 33)
- Need to convert for the HighSeq Reads:
- easy_install biopython
- git clone git://github.com/tanghaibao/jcvi.git
- export PYTHONPATH=/lib/python (which is the dir above jcvi)
- python -m jcvi.formats.fastq (Install missing packages)
Cleaning Single Reads:
- Sequences cleaned using trimReads by Haibao Tang: https://github.com/tanghaibao/trimReads/tree/
- Ran with supplied adapter sequence file:
>Adapter 4 TGACCA >Adapter 4 rc TGGTCA
- Command-line run:
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
Converting sequences
- python -m jcvi.formats.fastq convert (read help file, default conversion Sanger (code 33) to Illumina (code 64)
Other programs to clean sequences
- python -m jcvi.apps.baseclean trim fastqfile (single ended)
- python -m jcvi.apps.baseclean trim R1.fastq.gz R2.fastq.gz (paired ended)
keep sequences in single files (or two files for a pair of reads)
- Cat all the R1s together
- Cat all the R2s together
