Creosote Assembly

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/
  • Creosote First Run 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

Assembly using ABySS

By James S

ABySS requires paired end reads be marked in the names with "A" "B" or "1" "2" or "F" "R". The current read files didn't have this information, so I added an "A" to the end of every name in R1_all.pairs.fastq.gz using a perl one liner, and a "B" to the end of every name in R2_all.pairs.fastq.gz

Since this is paired end data I used the ABySS pair-end driver script 'abyss-pe'

Invocation:

• First attempt: abyss-pe k=55 n=10 -j2 name=creosote in="R1_all.pairs.fastq.gz R2_all.pairs.fastq.gz" se="merged_pairs.fastq.gz R1_all.frags.fastq.gz R2_all_frags.fastq.gz" <-- ABySS dies because the sequences in merged_pairs are not always the same length as the quality score strings

Note by Eric:  merged_pairs.fastq.gz is the merge of R1_all.pairs and R2_all.pairs.  This merged file was required by velvet.

• Second attempt (no single end data and added "A" and "B" to paired end reads): abyss-pe -j2 k=55 n=10 name=creosote in="R1_all_pairsA.fastq R2_all_pairsB.fastq"
• Third attempt (trying for longer contigs): abyss-pe -j2 k=64 n=5 name=creosote90 in="R1_all_pairsA.fastq R2_all_pairsB.fastq"