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=Moleculo Data Analysis Part 1= ==Background== *We are integrating Illumina (short read) and Moleculo (long read) data to assemble the oncomelania genome *The overall plan that we are using is: Test assembly of small Illumina dataset | V Validation of assembly (using assembly statistics and core genes search) | V Assembly of whole Illumina dataset | V Integration of Moleculo with Illumina reads | V Validation of hybrid assembly (assembly statistics, core genes search, assembly errors) *A more detailed plan can be found in: [[File:Moleculo_Data_Analysis_Plan_5_14_2013.docx]] ==Procedure== ===0_Illumina_Test_correction=== *Triton Folder: </projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/0_Illumina_Test_correction/> *In order to first start with the test assembly of small Illumina dataset, we first want to perform correction of reads and test whether correction will help improve assembly *We are using the correction software used in Assemblathon2 by SOAPdenovo: (found in </home/cjwei/software/SOAPdenovo2-src-r223/SOAPdenovoPipeline4BirdInAssemblathon2/> **kmerfreq **correct_error *Steps for error correction: 0) Need to gzip read files prior to error correction (can also use .gz files for assembly in SOAPdenovo) 1)Create read list for correction <reads2cor.lst>, which is just the file location of reads. If have paired read files, must put each file consecutively. /projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Illumina_raw/HB_gDNA200b_121224L1-4_R1_filtered.fastq.gz /projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Illumina_raw/HB_gDNA200b_121224L1-4_R2_filtered.fastq.gz /projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Illumina_raw/HB_gDNA2kb_NoIndex_L005_R1_001_filtered.fastq.gz /projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Illumina_raw/HB_gDNA2kb_NoIndex_L005_R2_001_filtered.fastq.gz 2) Run commands (need to run kmerfreq then correct_error scripts in order) <1_correction> *Parameters set based on those used in assemblathon2: **kmerfreq ***k=k-mer size, 13-27 (used 27) ***l=input read file list ***p=output prefix ***t=cores used (32 cores per node) ***i=set initial size of hash table (used 400000000) ***L=max read length (used 200 in order to ensure enough memory usuage set aside) **correct_error ***k=k-mer size, must be same as kmerfreq (used 27) ***l=low frequency cutoff (used 2) ***a=set whether remain all the data or not (used 1) ***e=set whether trim suspicious region at end of read according to Q-value or not (used 1) ***w=set whether trim error bases with Q-value <=2 instead of correct (used 1, yes) ***q=set quality threshold (35) ***t=number of cores (used 32) ***j=set whether convert read1 and read 2 corrected file into pair-end file (used 1, yes) /home/cjwei/software/SOAPdenovo2-src-r223/SOAPdenovoPipeline4BirdInAssemblathon2/kmerfreq -k 27 -l /projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1_Illumina_Test_Assembly/reads2cor.lst -p /projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1_Illumina_Test_Assembly/test_assembly -t 32 -i 400000000 -L 200 >/projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1_Illumina_Test_Assembly/kmerfreq.log /home/cjwei/software/SOAPdenovo2-src-r223/SOAPdenovoPipeline4BirdInAssemblathon2/correct_error -k 27 -l 2 -a 0 -e 1 -w 1 -q 35 -t 32 -j 1 /projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1_Illumina_Test_Assembly/test_assembly.freq.gz /projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1_Illumina_Test_Assembly/reads2cor.lst >/projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1_Illumina_Test_Assembly/correct.log 3) Output can be found in original raw read folder as .cor.pair files because program combines the paired read files into one </projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Illumina_raw/> HB_gDNA200b_121224L1-4_R1_filtered.fastq.gz.cor.pair HB_gDNA2kb_NoIndex_L005_R1_001_filtered.fastq.gz.cor.pair ===1a-c_Illumina_Test_Assembly=== *Continued on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/Microbiome/2013/2013-5-15>
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