Vicuna User Manual
Contents
1. fastq files npFqDir input folder for unpaired fastq files pFaDir input folder for paired fasta files np FaDir input folder for unpaired fastq files batchSize the max number of reads to be stored in the memory LibSize Upper Bound upper bound of fragment size min_output_contig_len min length of contigs to output outputDIR output directory path Table 2 Parameter settings for VicunAnalysis program General Parameters pFqDir npFqDir pFaDir npFaDir same as in Vicuna trim_log_file trim log output from Vicuna aln_file contig align output from Vicuna outputDIR output directory for VicunAnalysis Alignment output for particular region of interest num_region number of regions of interest for each region it specifies three tab delimitated fields contig number start and end positions on the contig Ufu_freq specify low frequency length polymorphism regions coverage compared to neighboring regions lfu_maz_freq max length for the low frequency length polymorphism region 6 Citing Vicuna Xiao Yang Patrick Charlebois Sante Gnerre Matthew G Coole Niall J Lennon Joshua Z Levin James Qu Elizabeth M Ryan Michael C Zody and Matthew R Henn 2012 De novo assembly of highly diverse viral populations in review 7 Contact If you have any question please email Xiao Yang xiaoyang broadinstitute org2. txt vicuna config Modify the parameters in file vicuna_config Note please uncomment the parameter names and there should be no space between the beginning of the line and the parameter name pFqDir the path of the input folder This folder should contain a multiple of two paired read fastq file ending with fq or fastq The user has to make sure that if there are more than 2 paired files when loaded into the memory two files that form pairs have to be loaded consecutively This can be achieved by naming a pair of read files to be 11 fq and i_2 fq for instance where i can be an integer or a string outputDIR the path of the output folder MSAFileName the path of the file stores the MSA of target genomes recommended to be used for samples rich in contamination Set this parameter to be seq viral analysis xyang programs Vicuna db hiv 1B algn for HIV seq viral analysis xyang programs Vicuna db wnv align for West Nile virus seq viral analysis xyang programs Vicuna db denv align for Dengue virus and seq viral analysis xyang programs Vicuna db lasvL align for L element of Las virus and seq viral analysis xyang programs Vicuna db lasvS align for S element of Las virus Run Vicuna OMP_NUM_THREADS 8 seq viral analysis xyang programs Vicuna bin vicuna omp v8 vicuna_config Note 1 you can bsub the above command for example bsub P ProjName o scre
3. Vicuna User Manual Xiao Yang Patrick Charlebois Michael C Zody and Matthew Henn Genome Sequencing and Analysis Program The Broad Institute of MIT and Harvard May 2012 Contents 1 General Description Quick Start for Broad Institute Users w Quick Start for external users dul Prereg isitel sos ad ae e Se ke aa ee ee Re A ee Re a 3 2 PTOCCUUTE ca se SR eRe e ee we Bw Ae ale Bae Se we ed NS Parameter Setting License i Citing Vicuna yl ontact 1 General Description Vicuna is a program for de novo consensus assembly of viral population It leverages efficient clus tering partitioning and alignment algorithms to make overlap layout consensus assembly strategy applicable to next gen datasets Vicuna has been used to assemble clinical HIV RSV West Nile and Dengue population and should be applicable to any other type of retrovirus sample Vicuna has been applied to both Illumina paired reads and 454 reads It should be directly applicable for Ion Torrent reads as well 2 Quick Start for Broad Institute Users 1 5 Use bash environment bash Export NCIB toolkit library path export LD_LIBRARY_PATH LD_LIBRARY_PATH seq viral analysis xyang programs Library ncbi_cxx 7_0_0 lib Copy Vicuna template config file to your local directory where user should specify the path parameter vicuna config cp seq viral analysis xyang programs Vicuna config miseq general
4. _ cools CURRENT e configure prefix path_to_install with optimization with mt with dll Note path_to_install needs to be specified by the user e make note this is gnu make e make install 2 Installation of Perl recent versions are recommended 3 g compiler recent versions are recommended 3 2 Procedure 1 Download the Vicuna package decompress and cd into the Vicuna folder 2 Switch to bash environment bash 3 Export NCIB toolkit library path Assuming you successfully installed NCBI Toolkit 7 0 0 in directory path then you should be able to find the library in directory path lib export LD_LIBRARY_PATH LD_LIBRARY_PATH path lib 4 Run Vicuna a Edit file Vicuna src Makefile set the parameter MYPATH to be path set the parameter COMPILER to be the path of the g compiler you are using you could use command which g to find out this information b Compile cd sre make cd Note e The executive file can be found in the Vicuna bin folder e By default the executive file is compiled with fopenmp flag and named vicuna omp v1 0 These settings can be changed by modifying the Makefile c Set basic parameters in file Vicuna config vicuna_config txt pFqDir the path of the input folder This folder should contain a multiple of two paired read fastq file ending with fq or fast
5. _log file the path of trim log file aln file the path of contig align file pFqDir the same as in Vicuna config vicuna config txt outputDIR the path to the output directory Instructions for setting other parameters are provided in the config file Execute bin vicunAnalysis config vanalysis_config txt The Output can be found in outputDIR folder contig fa contig output in fasta format contig lfv fasta contig output in fasta format contains low frequent variants 4 Parameter Setting Some high level explanations for Vicuna 1 Vicuna handles both paired and unpaired read files containing reads with variable length Currently paired reads have to be present as part of the input see section on how to handle only unpaired reads and only fastq and fasta format are handled This may change in newer version 2 When reading files from a folder directory Vicuna assumes two paired read files are read in consecutively In order to achieve this you can give the paired files with the same prefix but different suffix For example in a folder we have two sets of paired files a b and c d then we can assign the following names to each of these files a 1 p1 fastq b 1 p2 fastq c 2 pl fastq d 2 p2 fastq These files should then be read in comforming alphabetical order 3 Read ID rID assignment Each read is assigned with a unique ID with the following r
6. en_output txt q hour W 4 00 R rusage mem 6 n 2 8 R span hosts 1 vicuna vicuna_config Please replace the parameters between 2 you can change 8 to the number of cores CPUs you wish to use The Output can be found in outputDIR folder trim log specifies the trimming history of each read contig align specifies the read alignment to the consensus generated 6 Parse Vicuna output using analysis script a Copy the analysis template config file to your local directory where the user should specify the path parameter analysis_config cp seq viral analysis xyang programs VicunAnalysis config txt analysis_config b Modify parameters in file analysis_config trim_log_file the path of trim log file aln file the path of contig align file pF qDir the same as in vicuna_config outputDIR the path to the output directory Instructions for setting other parameters are provided in the config file c Run analysis seq viral analysis xyang programs VicunAnalysis vicunAnalysis analysis_config The Output can be found in outputDIR folder contig fa contig output in fasta format contig lfv fasta contig output in fasta format contains low frequent variants 3 Quick Start for external users 3 1 Pre requisite 1 Installation of NCBI Toolkit 7 0 0 download link ftp ftp ncbi nih gov toolbox ncbi
7. he bin i r is assigned to bin 7 rMapFileName if specified two output files are created rMapFileName record txt records only mapped rIDs rMapFileName is a tab delimited file each line has three entries 1 rID 2 BinID and 3 isPaired specifying if the paired end of rID is assigned Contig Construction Validation and Extension w1 kmer size for the first iteration of min hash w2 kmer size for the second iteration of min hash Divergence max of divergence between read amp consensus during contig validation maz_read_overhang number of base pairs that can be ignored towards either end of a read during contig validation This number accounts for insufficient trimming PCR artifacts sequencing errors etc maz_contig_overhang max length of unreliable region in either end of the consensus to be tolerated during contig merging min_perc_polymorphism min frequency of length polymorphic region to be considered to be part of a contig mazx_variant_len max length of any variant that will be removed b4 aligning two contigs seed_kmer_len seed kmer length for computing overlap between two contigs min_contig_overlap min length of overlaps between two contigs for them to be merged min_contig_links min number of paired links for attempting to merge two contigs min_identity min similarity to merge two contigs General Parameters pF qDir input folder for paired
8. ile of the n contig e contig lfv fasta the fasta format of consensus sequences retaining any low frequent length polymorphisms The consensus in this file corresponds to contig align e contig fa consensus sequences without low frequent length polymorphisms See Table 1 for Vicuna program and Table 2 for VicunAnalysis program 5 License Please refer to license folder Table 1 Parameter settings for Vicuna program Trimming remove known primer sequences vectorFileName the path of the Fasta file that stores primer vector sequence s to be removed from each read minM Size if the suffix or prefix of a read matches any substring of some primer with length gt minMSize the matching part is trimmed minInternalM Size if an internal substring of a read matches any substring of some primer with length gt minInter nalM Size the full read is trimmed maz OverhangSize max number of neglect able bases for a substring in a read to be considered as suffix or prefix minReadSize the min length of read to be retained before or after trimming Profiling identify target alike reads MSAFileName the path of the Fasta file storing MSA of previously assembled target genomes binNumber the number of bins the MSA is divided into kmerLength kmer length marHD max Hamming distance tolerated between two kmers minSpan if gt minSpan positions of r is covered by kmers from t
9. q The user has to make sure that if there are more than 2 paired files when loaded into the memory two files that form pairs have to be loaded consecutively This can be achieved by naming a pair of read files to be i_1 fq and i_2 fq for instance where i can be an integer or a string outputDIR the path of the output folder MSAFileName the path of the file stores the MSA of target genomes rec ommended to be used for samples rich in contamination We provided this file for HIV Vicuna db hiv 1B fa West Nile Vicuna db wnv fa Dengue Vicuna db denv fa and Las virus Vicuna db lasvL fa for L element Vi cuna db lasvS fa for S element d For advanced users instructions for setting each parameter are provided in the file Vicuna config vicuna_config txt Execute Vicuna OMP_NUM_THREADS n bin vicuna config vicuna config txt n is the number of cpus you would like to use e g 4 The Output can be found in outputDIR folder trim log specifies the trimming history of each read contig align specifies the read alignment to the consensus generated 5 Parsing the output of Vicuna using vicuna analysis program a Compile cd scripts VicunAnalysis make clean make all cd The output will be written as Vicuna bin vicunAnalysis Set basic parameters in the config file Vicuna config vanalysis_config txt trim
10. ules 1 the first read is assigned with ID 0 2 for paired reads r r2 rl Da rID 1 if ra is read in after r 3 if ra is read in right after r if both are in the same file then rI D rID 1 if this file is unpaired otherwise rf D rI D 2 if they are in different files then rf Dz rI D 1 if the two files are not paired 4 For any calculation e g generating consensus of contig reads are loaded in batches with user specified batch size This controls memory usage in case when input consists of a large number of reads 5 Output files from Vicuna e trim log record the trimming information e contig align record contig alignment information e contig lfv fasta the fasta format of consensus sequences retaining any low frequent length polymorphisms The consensus in this file corresponds to contig align e contig fasta consensus sequences without low frequent length polymorphisms Some high level explanations for VicunAnalysis 1 Using VicunAnalysis you can print out alignments of specific region of a specific contig of interest Particularly if you are interested in length polymorphic regions or low coverage regions 2 VicunAnalysis can output the raw consensus sequences corresponding to the MSA of each contig output from Vicuna or you can remove low frequent polymorphisms from the consen sus 3 Output files from VicunAnalysis e contig n txt record the prof
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