SPANDx Manual_v2.3
Contents
1. User Manual Manual version 2 3 last modified 28Jul15 For the latest version of SPANDx and its associated user manual please visit either of the following SPANDx websites http sourceforge net projects spandx or https github com dsarov SPANDx CONTENTS Introduction and Description Synopsis Commands and Options Installation and Requirements Interpreting the Outputs Examples Authors and Citation References INTRODUCTION AND DESCRIPTION SPANDXx Synergised Pipeline for Analysis of Next generation sequencing Data in Linux is a comparative genomics pipeline designed to greatly simplify the identification of genetic variants SNPs insertions deletions indels and large gt 200bp deletions from medium to large sized haploid next generation re sequencing NGS datasets SPANDx can currently process several NGS data inputs including paired and single end data from the Illumina MiSeq HiSeq and GA platforms single end data from the Life Technologies lon Personal Genome Machine PGM and single end Roche 454 data SPANDx integrates the following validated bioinformatics tools for start to finish sequence analysis of raw NGS data using a single command Burrows Wheeler Aligner BWA 1 2 for alignment of short i e Illumina and PGM or long i e 454 NGS read data BWA is downloadable from http bio bwa sourceforge net SPANDx does not currently support BWA versions later than 0 6 2 SAMtools 3 and Pica2. PE w INT Optional The w switch specifies the window size in base pairs used by BEDTools to analyse whole genome alignment coverage i e locus presence absence 1000 INSTALLATION AND REQUIREMENTS SPANDXx is written in Bash and will run on most Linux installations For parallelisation SPANDx can utilise PBS SGE or SLURM resource managers SPANDx has been tested on GNU Bash version 3 2 25 1 release and GNU Bash version 4 1 2 1 release both on x86 64 redhat linux gnu with Java v1 7 0 55 and v1 7 0 71 The 2 7 version of SPANDx downloadable here http sourceforge net projects spandx or here https github com dsarov SPANDx has been tested using PBS TORQUE v2 5 13 SGE v6 2u5p3 and SLURM v14 11 Dependency versions tested in SPANDx v2 7 are BWA v0 6 2 SAMtools v0 1 19 Picard v1 105 the Genome Analysis Toolkit v3 0 BEDTools v2 18 2 SnpEff v4 1 VCFtools v0 1 11 and tabix v0 2 6 SPANDx does not currently support BWA versions later than 0 6 2 For parallelisation SPANDx requires PBS SGE or SLURM to submit jobs to the cluster If you do not have one of these system setups SPANDx cannot be run in parallel Please contact us if you are using a different Linux resource scheduler setup and are willing to trial and error SPANDx on your system SPANDx should work from any installation directory but has been most extensively tested in home user bin To install SPANDx gunzip and untar the program usually with the command
3. BEDCov window size of 500bp instead of the default 1000bp desired using the same reference genome as above home user bin SPANDx SPANDx sh r Hi 86 028NP o Hi t PGM p SE w 500 Paired end Illumina 1 3 reads annotated genome available required for the reference genome Hi 86 028NP fasta with a single strain Hi 00345 for alignment and variant calling No SNP indel matrices required home user bin SPANDx SPANDx sh r Hi 86 028NP o Hi a yes v Haemophilus influenzae 86 028NP uid58093 t Illumina old g Hi 00345 mGWAS generation of input data compatible with PLINK Running PLINK for mGWAS analysis across antibiotic resistant strains vs antibiotic sensitive strains 14 home user bin SPANDx GeneratePlink sh i inGroup antibiotic resistant txt o OutGroup antibiotic sensitive txt Running PLINK for mGWAS analysis as above but changing locus presence absence cutoffs for customised outputs default is 0 9 home user bin SPANDx GeneratePlink sh i inGroup antibiotic resistant txt o OutGroup antibiotic sensitive txt 6 0 95 15 AUTHORS AND CITATION SPANDx was developed by Dr Derek Sarovich and Dr Erin Price from Menzies School of Health Research Darwin NT 0810 Australia Derek s homepage http www menzies edu au page Our People Researchers Derek Sarovich Erin s homepage http www menzies edu au page Our People Researchers Erin Price If you find an error or bug please contact Derek or Erin at mshr bioin
4. NC 012967 590 NC 012967 620 NC 012967 632 NC 012967 659 NC 012967 668 NC 012967 689 na naa naa 440 naa nao nnn nao nao naa nao nao naa N40 NAANANEPANADANANGAA ANDAANANDANADANNNAYS ANPAANNENADANNANGAA ANDANANDANDANANADYS NANANAPANAPANNGAA ANDANANEPANDANANDA NAANNAPOANY PAHADOHSA NAANANDANAANNNAA NAANANDANDANNANAA ABPANNNEPANADANNANGA NAANANPANAANNNGAA ANAANANPANDANNANGAA NAANANPANDANNANGAA NAANANEPANDANANGAA NAANANEPANAANANGAA NAANANPANAANANGAA NAANANPANDANNANGAA Ortho SNP matrix RAxML nex is a RAxML and PHYLIP importable version of the Ortho SNP matrix RAxML nex file Note that for compatibility with PHYLIP taxa names must have exactly 10 characters including spaces otherwise Ortho SNP matrix RAxML nex will not be recognised as a valid PHYLIP file SPANDx does not automatically rename taxa to meet this PHYLIP requirement Merged SNP indel matrices In certain instances particularly in outbreak studies where only closely related strains are being examined and there are few genetic variants it may be desirable to merge SNP and indel variants prior to phylogenetic analysis for increased resolution We have recently shown that this approach can provide more robust phylogenetic correlation with epidemiological data than constructing a phylogeny based on SNPs alone 12 To merge SNP and indel variants into a separate output file run the MergeSnpIndel sh script while in the PWD Outputs Comparative dire
5. Phylo snps and perform error correction using the bam and bai files contained in PWD Phylo bams to construct an orthologous SNP matrix which will be output to SPWD Phylo out Before running this module please check that all vc files located within SPWD Phylo snps match the alignment files within SPWD Phylo bams and 6 that all bam files contain their accompanying bai index file This feature mitigates the need to re run previous analyses from scratch and is useful for combining data generated from multiple SPANDx runs e g from different sequencing technologies into a single orthologous SNP matrix all t STR Optional The t switch specifies the sequence technology used and must be one of the following 1 lumina Illumina old 454 or PGM By default SPANDx expects Illumina reads with Phred 33 read quality encoding which is standard as of v1 8 To specify NGS reads generated by the older Illumina format i e Phred 64 use t Illumina old If the analysis mode is switched to 454 or PGM SPANDx will use the BWA SW algorithm of BWA for read alignment and thus will expect reads to be in single end strain 1 sequence fastq gz format with Phred 33 quality encoding Zl lumina p STR Optional The p switch specifies the pairing of reads and must be either PE or SE By default SPANDx expects reads to be paired If reads are single end must be set to SE Currently SPANDx does not support paired end 454 or PGM data
6. with v1 8 quality encoding If your data are in this format the only required switch is r to specify the reference sequence prefix If another NGS data format is being analysed please specify this format using the t and if single end the p switch es By default SPANDx will construct a locus presence absence matrix but will not construct orthologous SNP or indel matrices nor will it perform variant annotation The m matrix and i indel switches are required for orthologous SNP and indel matrix creation respectively The a annotate and v reference name for variant identification switches are both required for variant annotation SPANDx cannot process multiple NGS formats e g single end and paired end Illumina in a single run If multiple NGS formats are to be analysed please create separate analysis directories and run SPANDx specifically for each NGS format These data can be merged for downstream analysis See the s description for more information Prior to running SPANDx sh both the reference in fasta format and NGS files in fastgq gz format are required to be in your analysis directory SPANDx expects all NGS reads to be in the sequence analysis directory i e the present working directory and by default all NGS reads within the sequence analysis directory will be processed Before running SPANDx make sure the NGS read files conform to the following format regardless of the sequencing technology used str
7. L Cardle L Shaw PD Marshall D 2013 Using Tablet for visual exploration of second generation sequencing data Briefings in Bioinformatics 14 193 202 McRobb E Sarovich DS Price EP Kaestli M Mayo M Keim P Currie BJ 2015 Tracing melioidosis back to the source using whole genome sequencing to investigate an outbreak originating from a contaminated domestic water supply J Clin Microbiol 53 1144 1148 17
8. STREAM DING LOW MODIFIER MODIFIER SILENT 130 gspG 71 gspK 9 gspK ctC ctT LOW SILENT LOW SILENT 1 16789 A G G G 1 17077 T C C 0222 0222 SYNONYMOUS CODING SYNONYMOUS CODING caA caG gtT gtC Q133 V229 gspK gspK BPSL0059 BPSL0059 022 0222 LOW SILENT LOW SILENT 1_65151 C T T 1_65664 A G G G SYNONYMOUS_CODING SYNONYMOUS_CODING gaG gaA E199 atT atC 128 LOW LOW SILENT SILENT BPSLO319 BPSLO320 1 340115C Li T T 1 340333 T C C E 0222 0222 SYNONYMOUS CODING SYNONYMOUS CODING gcG gcA A6 gcA gcG A235 N B SNPs indels represented with are an ambiguous call and should be interpreted with caution SNPs indels represented with do not pass the depth filters and are likely in deleted regions SPANDx can repeat the variant filtering steps without repeating the alignment and data processing steps To use this behaviour remove the relevant snps PASS and indels PAss files from the SPWD Outputs SNPs indels PASS directory and the relevant snps AMB and indel Amp files from the SPWD Outputs SNPs indels FAIL directory change the GATK config file to the desired parameters and re run SPANDx NB SPANDx will only re filter the variants with altered parameters for those strain s that have been removed from the Output directories Orthologous SNP matrices for phylogenetic analyses Two separate SNP matrix files are generated by SPANDx These matrices are ou
9. ain 1 sequence fastq gz and strain 2 sequence fastq gz for paired end reads or strain 1 sequence fastq gz for single end reads See screenshot below for correctly formatted paired end input files Jj SPANDx input amp v e fe amp m ZB findFiles Fa Eig Download 3g 2 Edit 9 m4 Lz Properties EY 7 V T Name Bt Size Changed Rights Owner 2 i 12 04 2015 7 34 23 PM TWXI Xr X eprice al REL6O6 fasta 12 04 2015 7 35 41 PM rWXTWXTWX eprice E M300 1 sequence fastq gz 12 04 2015 7 35 41 PM rWXrWwxrwx eprice ie M300 2 sequence fastq gz 12 04 2015 7 35 41 PM TWXTWXTWX price E M331_1_sequence fastq gz 12 04 2015 7 35 41 PM Twxrwxrwx eprice i M331_2_sequence fastq gz 12 04 2015 7 35 41 PM Twxrwxrwx eprice B M379 1 sequence fastq gz 12 04 2015 7 35 41 PM Twxrwxrwx eprice fe M379_2_sequence fastq gz 12 04 2015 7 35 41 PM rwxrwxrwx eprice ie M615_1_sequence fastq gz 12 04 2015 7 35 41 PM Twxrwxrwx eprice E M615_2_sequence fastq gz 12 04 2015 7 35 41 PM Twxrwxrwx eprice E M617 1 sequence fastq gz 12 04 2015 7 35 41 PM Twxrwxrwx eprice B M617 2 sequence fastq gz 12 04 2015 7 35 41 PM Twxrwxrwx eprice n1 M618 1 sequence fastq gz 12 04 2015 7 35 41 PM Twxrwxrwx eprice Ee M618_2_sequence fastq gz 12 04 2015 7 35 41 PM Twxrwxrwx price B M619 1 sequence fastq gz 12 04 2015 7 35 41 PM TWXTWXTWX eprice ie M619_2_sequence fastq gz 12 04 2015 7 35 41 PM Twxrwxrwx eprice ie M620 1 sequence fastq gz 12 04 2015 7 35 41 PM Twxrwxrwx
10. ctory The MergeSnpIndel sh script will merge and reformat the data in the Ortho SNP matrix nex and indel matrix nex files The output file is indel SNP matrix nex This file is directly importable into PAUP Below is a screen capture of this file nexus begin data dimensions ma nchar 107451 format symbols 01 gap datatype standard transpose taxlabels REL606 308 M331 M379 M615 M617 M618 M619 M620 M632 REL10938 REL1164A REL2179A REL4536A REL607 REL7177A REL8593A matrix nc_012967_58 00000000010000000 nc_012967_64400000000010000000 nc_012967_67 00000010000000000 NC 012906717101010000000000000 NC 012967 39201010001100000000 NC 012906743700000010000000000 Nc 0129067 46000000010000000000 NC 0129067 47300001010000000000 NC 012967 47900001010000000000 nC_012967_5090 0000010000000000 Nc_012967_536 0 0100000000000000 NC 0129067 558800101010000000000 NC 012967 58701010001100000000 NC 012967 55000001010000000000 NC 012967 62000101110010000000 NC 0129067 632200100000000000000 NC 0129067 65900001000000000000 NC 012967 66800100000010000000 12 Microbial genome wide association studies mGWAS The main comparative outputs of SPANDx SNPs indels and presence absence matrices in SPWD Outputs Comparative can be used as input files for mGWAS As of version 2 6 SPANDx is distributed with GeneratePlink sh The GeneratePlink sh script requires two input files an ingroup txt file and an outgroup txt file The ingroup txt file should c
11. e SPANDx workflow is shown below Raw DNA sequencing read s fastq gz KEY Read alignment against input fies reference BWA Export of unaligned Individual reads bam genome analysis Conversion to bam romeo reri format SAM Tools Comparative 3 Removal of optical duplicates Picard Local realignment of Optional step high mismatch regions GATK gea p absence matrix SPANDx MS Excel interrogate ambiguou variants GATK enetic analysis PAUP Rei PHYLIP RAxML i emove non orthologous and low quality SNPs matrix nex f yed Merge annotated variants i SNPEff SPANDx SYNOPSIS SPANDx r exact reference name xcluding fasta extension parameters optional o organism m generate SNP matrix yes no i generate indel matrix yes no a include annotation yes no v referenc fil for variant annotation name must exactly match the SnpEff database name which can be found in the snpEff config file s specify read prefix to run single strain set to none to construct a SNP matrix from a previous analysis or leave as default to process all reads sequencing platform i e Illumina Illumina old 454 PGM p pairing of reads i e paired end or single end PE SE w BEDTools window size in base pairs COMMANDS AND OPTIONS SPANDx shisthe only script that needs to be run to obtain data outputs SPANDx by default expects paired end Illumina data
12. e compiled with the SPANDx package GATK needs to be downloaded and installed separately due to Broad Institute licencing restrictions PLINK has not been included in the SPANDx bundle yet but will be included in future versions The dependency binaries have been compiled for an x86 64 Linux system If you have different system architecture you will need to install SPANDx dependencies yourself Novel comparative genomic features of SPANDx include Merged orthologous SNP and indel matrices that greatly simplify variant visualisation for comparative genomic analyses PAUP PHYLIP and RAxML compatible nex orthologous SNP matrix files for downstream phylogenetic analyses PHYLIP and RAxML are freely available programs downloadable from http evolution genetics washington edu phylip html and http www exelixis lab org As of July 2015 PAUP is now open source and will be frequently updated by the developer over the coming months Please use Google to find the latest version Locus presence absence matrices from BEDTools outputs that enable simple visualisation and comparative genomic determination of 1 the core genome and 2 variable genetic loci including deleted regions brought about by reductive evolution Merged annotated SNP and indel matrices for fast and simple genetic characterisation of variants NB The user must provide SPANDx with the SnpEff annotated reference genome information for variant annotati
13. eprice SARRFHFKRHRRHSRKRKRRRSHRE Options r STR Required Specifies the reference genome file excluding the fasta extension The r SWitch is the only mandatory switch needed for SPANDx to function Additional switches are required to modify the default behaviour of SPANDx and sequencing technology needs to be specified if your data are not paired end Illumina data with v1 8 quality scores The reference file is required to be in fasta format and should conform to the standard FASTA specification as detailed here http www ncbi nlm nih gov BLAST blastcgihelp shtml IUPAC codes are not supported by some programs incorporated in SPANDx and must be avoided For compatibility with the annotation module of SPANDx the chromosome names for the reference genome must match those used by SnpEff This nomenclature can be found in the snpEff config file which is generated upon SnpEff installation or automatically with the full SPANDx installation In addition the fasta reference file must not contain any blank lines 0 STR Optional Specifies the organism under analysis The o parameter is used in naming the read group headers in the SAM and BAM files Spaces and special characters may have unexpected behaviour and should be avoided Haploid m yes no Optional The m switch is used to create a matrix with all orthologous SNP variants identified in the analysis Non orthologous SNPs are excluded Output nex f
14. erated by SAMTools after BWA alignment The unaligned reads can be found in SPWD strain unique unmapped bam It is anticipated that future versions of SPANDx will include an option for automated assembly of these unaligned reads Alignment files Alignment files generated with SPANDx are in bam format and are found in SPWD strain unique strain realigned bam If visualisation of the alignment is desired these files can be easily viewed in an alignment viewer our favourite is Tablet 11 downloadable from http ics hutton ac uk tablet Whole genome coverage a k a locus presence absence Following assessment of whole genome coverage by BEDTools SPANDx provides a combined BEDcov matrix for all analysed genomes in PWD Outputs Comparative Bedcov merge txt This file lists the BEDTools windows or NGS read coverage for each strain based on the reference sequence and ranges from O 096 read coverage across the window to 1 100 coverage across the window The BEDcov matrix can be imported into Microsoft Excel for easier visualisation and manipulation Bedcov merge txt is a useful file for identifying the core genome of a given dataset and for identifying variable genetic regions among strains The bp resolution of this output can be modified by changing the w switch e g 100 cf the default 1000 We suggest changing to 100 for microbial GWAS analysis in PLINK to increase sensitivity and leaving as default for all other analy
15. formatics 2 gmail com Please include a detailed description of the error you encountered the operating system you used and what happened to cause the error In addition please send the appropriate log files with the description Derek can be followed on Twitter DerekSarovich https twitter com dereksarovich Any feedback regarding SPANDx is most welcome If you used SPANDx and found it useful for your research please cite it Sarovich DS and Price EP 2014 SPANDx a genomics pipeline for comparative analysis of large haploid whole genome re sequencing datasets BMC Res Notes 7 618 16 REFERENCES 10 11 12 Li H Durbin R 2009 Fast and accurate short read alignment with Burrows Wheeler transform Bioinformatics 25 1754 1760 Li H Durbin R 2010 Fast and accurate long read alignment with Burrows Wheeler transform Bioinformatics 26 589 595 Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth G Abecasis G Durbin R Subgroup GPDP 2009 The Sequence Alignment Map format and SAMtools Bioinformatics 25 2078 2079 McKenna A Hanna M Banks E Sivachenko A Cibulskis K Kernytsky A Garimella K Altshuler D Gabriel S Daly M DePristo MA 2010 The Genome Analysis Toolkit a MapReduce framework for analyzing next generation DNA sequencing data Genome Research 20 1297 1303 DePristo MA Banks E Poplin R Garimella KV Maguire JR Hartl C Philippakis AA del Angel G Rivas MA Hanna M McKenna A Fenne
16. iles can be directly imported into PAUP PHYLIP or RAxML for phylogenetic analysis By default this behaviour is switched off no i yes no Optional The i switch is used to create a matrix with all orthologous indel variants identified in the analysis Non orthologous indels are excluded By default this behaviour is switched off no a yes no Optional The a switch is used to perform annotation of the variant files By default this behaviour is switched off If annotation is switched on the v switch must also be specified no v STR Optional required if a is set to yes The v switch is used to specify the reference genome that SnpEff will use to annotate variants The string used for this variable must match one of the genomes contained within the SnpEff config file Additionally chromosome names in the reference file must match those contained within the SnpEff program Please refer to the SnpEff manual which can be found here http snpeff sourceforge net SnpEff manual html for more information null s STR Optional The s switch is used to flag a single genome for analysis If s is set to none SPANDx will not perform individual analysis of any NGS read files in the current directory Instead SPANDx will move to the comparative genomics section of the pipeline see SPANDx workflow above and assume all individual genome analysis has already been completed SPANDx will then merge all VCF files contained in SPWD
17. ll TJ Kernytsky AM Sivachenko AY Cibulskis K Gabriel SB Altshuler D Daly MJ 2011 A framework for variation discovery and genotyping using next generation DNA sequencing data Nat Genetics 43 491 498 Van der Auwera GA Carneiro MO Hartl C Poplin R Del Angel G Levy Moonshine A Jordan T Shakir K Roazen D Thibault J Banks E Garimella KV Altshuler D Gabriel S DePristo MA 2013 From FastQ data to high confidence variant calls the Genome Analysis Toolkit best practices pipeline Curr Protoc Bioinformatics 11 11 10 11 11 10 33 Danecek P Auton A Abecasis G Albers CA Banks E DePristo MA Handsaker RE Lunter G Marth GT Sherry ST McVean G Durbin R Genomes Project Analysis G 2011 The variant call format and VCFtools Bioinformatics 27 2156 2158 Quinlan AR Hall IM 2010 BEDTools a flexible suite of utilities for comparing genomic features Bioinformatics 26 841 842 Cingolani P Platts A Wang le L Coon M Nguyen T Wang L Land SJ Lu X Ruden DM 2012 A program for annotating and predicting the effects of single nucleotide polymorphisms SnpEff SNPs in the genome of Drosophila melanogaster strain w1118 iso 2 iso 3 Fly Austin 6 80 92 Purcell S Neale B Todd Brown K Thomas L Ferreira MA Bender D Maller J Sklar P de Bakker PI Daly MJ Sham PC 2007 PLINK a tool set for whole genome association and population based linkage analyses Am J Hum Genet 81 559 575 Milne I Stephen G Bayer M Cock PJ Pritchard
18. matically installed with VCFtools in the SPANDx config file PERL5 libraries are required for correct functioning of VCFTools Make sure that the location of tabix and bgzip dependencies of VCFtools are specified in your PATH variable Some of these programs have additional dependencies that are required for them to function properly e g Java Please refer to the appropriate manuals or system administrator for installation of these utilities if they are not already on your Linux system SPANDx customisation Resource manager Depending on your cluster environment the scheduler config file may need to be changed By default SPANDx will expect the resource manager to be PBS IMPORTANT If you are using SGE please modify the SCHEDULER variable to SGE If you are using SLURM please modify the SCHEDULER variable to SLURM If no resource manager is available please modify the SCHEDULER variable to NONE This file will configure the operation of the resource manager By default all commands request one node and 12 hours of wall time you will not be sent mail and standard output is merged with standard error These settings can be changed if your job needs more time to complete or if you want e mail notifications of job completion Variant calling One advantage of SPANDx over other tools is that GATK variant calling parameters are already specified These parameters have been tested across NGS data for several bacterial species generated
19. on different NGS platforms Therefore the default settings should work well for most haploid genome projects If desired users can customise the SPANDx variant filtering parameters by altering the GATK config file All filtering steps used in GATK can be customised using this configuration file Note that these variables must conform to JEXL specifications SPANDXx variant calling has been optimised for bacterial genomes and therefore may behave differently for other haploid organisms If in doubt outputs should be verified with e g wet lab analysis of variants SPANDx is currently not configured to analyse diploid or polyploid genomes The following parameters can be customised to change the variant filtering behaviour for both SNPs and indels if required below are the default SPANDx settings CLUSTER SNP 3 for SNPs only CLUSTER WINDOW SNP 10 for SNPs only MLEAF 0 95 QD 10 0 MQ 30 0 FS 10 0 HAPLO 20 0 QUAL 30 0 OW DEPTH 2 variants with less than the average coverage divided by LOW DEPTH will fail filtering If this value is set at the default of 2 regions with less than half the average depth will fail and thus will be filtered out HIGH DEPTH 3 a value of 3 means that regions with more than three times the average coverage of the entire genome will fail and thus will be filtered out 9 INTERPRETING THE OUTPUTS Unaligned reads A bam file of the unaligned reads is gen
20. on to work The term orthologous refers to genetic loci that make up the core genome SNPs or indels residing in genetic loci that are missing in one or more genomes are excluded If these variants are required they can be found in the individual filtered vc output files Unlike many other comparative bioinformatics tools SPANDx does not require pre assembled genomes In addition the default settings for variant calling using Illumina lon PGM and 454 data have already been optimised and do not require the user to specify these settings although these settings can be customised if required SPANDx has been purposely written for systems that utilise Portable Batch System PBS Sun Grid Engine SGE i e qsub or Simple Linux Utility for Resource Management SLURM i e sbatch to enable task parallelisation greatly reducing turn around time of datasets comprising tens through to thousands of haploid genomes using a single command As of version 2 7 SPANDx will also run on systems without a resource manager but will not run in parallel Currently there is no support for SPANDx on other resource management systems e g LSF due to the unavailability of such systems in our laboratory but compatibility with resource managers can be addressed if required Please email us at mshr bioinformatics 9 gmail com if you require a specific resource manager compatible version of SPANDx and are willing to test it on your system Th
21. ontain a list of the strains of interest e g antibiotic resistant strains and the outgroup txt file should contain a list of all strains lacking the genotype or phenotype of interest e g antibiotic sensitive strains Although larger taxon numbers in the ingroup and outgroup files will increase the statistical power of mGWAS it is better to only include relevant strains i e do not include strains that have not yet been characterised The ingroup txt and outgroup txt files must include only one strain per line and must be in UTF 8 text file format do not save in other formats This script will generate ped and map files for SNPs and presence absence loci and for indels if these were identified in the initial analyses The ped and map files can be directly imported into PLINK For more information on GWAS and how to run PLINK please refer to the PLINK website http pngu mgh harvard edu purcell plink Log files By default both the standard error and standard output are merged into a single log file Almost all commands in SPANDx are captured in log files If an error occurs the log files are a good first place to look If you wish to minimise the amount of log files that SPANDx generates you can change the ERROR OUTPUT variable PBS to n or the ERROR OUTPUT SGE SGE to no in the scheduler config file Please note that this feature only works when using PBS or SGE resource handlers 13 EXAMPLES The simples
22. rd as yet unpublished for alignment manipulation and filtering These programs can be downloaded from http samtools sourceforge net and http picard sourceforge net respectively Genome Analysis Tool Kit GATK 4 6 for base quality score recalibration realignment of regions with low mapping quality duplicate removal identification of SNPs and indels and filtering the variant call format VCF file generated from the alignment process GATK can be downloaded from http www broadinstitute org gatk VCFtools 7 for manipulation of VCF files VCFtools is downloadable from http vcftools sourceforge net All file outputs from SPANDx are in standardised VCFv4 1 format tabix and bgzip are VCFtools dependences required for handling vc files and can be downloaded here http sourceforge net projects samtools files tabix BEDTools 8 and specifically the coverageBED module for identification of locus presence absence across each genome of interest based on the reference sequence This tool is useful for identifying larger scale approx 100bp or larger deletions BEDTools can be downloaded from https github com arq5x bedtools2 SnpEff 9 for annotation of SNP and indel variants SnpEff can be downloaded from here http snpeff sourceforge net PLINK 10 for microbial genome wide association studies mGWAS All of the above dependencies with the exception of GATK and PLINK come pre bundled and pr
23. ses Variants SNPs and indels are output from SPANDx analysis into two locations 1 PWD Outputs SNPs indels PASS which contains both SNP and indel variants that have passed the filters specified in the GA TK config file see Variant calling above for details of the default filters and 2 SPWD Outputs SNPs indels FAIL Which contains SNPs and indels that have failed filtering parameters If annotation is switched on annotated variants will be output to SPWD strain unique annotated Annotated SNP and indel outputs will be generated for each genome under analysis In addition if both annotation and comparative analysis is switched on a yes and m yes annotated merged SNP and indel matrices are generated for all genomes under analysis These files are found in S PWD Outputs Comparative and are called All indels annotated txt and A11 SNPs annotated txt These are tab delimited text files that can be easily imported into Excel as shown in the screenshot below Note that the binary column will need to be specified as Text only due to the character string containing O The binary column is a representation of the SNP indel pattern at that specific location which can be useful for filtering 10 Location K96243 MSHR5662 MSHR5667 MSHR5670 Binary code Effect Impact Functional_Class Codon change Amino Acid change Gene name 1 14099 C Li T L 1_16320 A G G G 1_16382 G C C E 0222 0222 0222 SYNONYMOUS CO UPSTREAM UP
24. t way to run SPANDx is if your reads are in paired end Illumina format and follow the naming convention of strain 1 sequence fastq gz and strain 2 sequence fastq gz SPANDx can then be run by simply specifying the reference fasta genome prefix All read files in the current directory will be processed although a SNP or indel matrix will not be constructed nor will variant annotation be performed unless specified No frills SPANDx command for basic Illumina 1 84 analysis home user bin SPANDx SPANDx sh r reference If other SPANDx features are required or reads other than Illumina v1 8 are used these features will need to be specified as per the examples below Paired end Illumina 1 8 reads SNP matrix required no annotated genome available required home user bin SPANDx SPANDx sh r referenc m yes To include an annotation for the above example home user bin SPANDx SPANDx sh r referenc a yes m yes v ref genome in SnpEff database Paired end Illumina 1 8 reads indel matrix required no annotated genome available required home user bin SPANDx SPANDx sh r referenc i yes m yes Paired end Illumina 1 3 reads SNP and indel matrices required annotated reference genome Hi 86 028NP fasta available home user bin SPANDx SPANDx sh r Hi 86 028NP o Hi m yes i yes a yes v Haemophilus influenzae 86 028NP uid58093 t Illumina old Single end lon PGM reads SNP indel matrices and annotation not required
25. tar xvfz SPANDx full tar gz in your bin directory Optionally SPANDx now has its own Github page meaning it can be installed directly using the following command git cione https github com dsarov SPANDx IMPORTANT following extraction of the script files the SPANDx config file will need to be modified to contain the location of the SPANDx installation If your system uses a proxy to access the internet please modify the JAVA PROXY variable in SPANDx config For default SPANDx behaviour no other settings should need to be modified In addition you will also need to download and install GATK and either place the GenomeAnalysisTK jar file renamed without version numbers in the SPANDx installation directory or specify the install path in the SPANDx config file For users who don t have x86 64 systems You will need to download and install SPANDx dependencies yourself as only x86 64 binaries are included in the SPANDx distribution Following extraction of the script files the SPANDx config file will need to be modified to contain the absolute paths of each dependency i e BWA SAMTools Picard GATK BEDTools SnpEff VCFtools and tabix installations see Introduction and Description above for web links to these free third party programs If a dependency not found error occurs please check the installation path and the location specified in the SPANDx config file Please make sure to specify the location of the PERLS libraries auto
26. tput in PWD Outputs Comparative and are named Ortho SNP matrix RAxML nex and Ortho SNP matrix nex The Ortho SNP matrix files generated by SPANDx exclude SNPs that are low quality that are in non orthologous regions and that are tri or tetra allelic in nature Non orthologous SNPs should not be used for phylogenetic reconstruction In addition filtering for tri and tetra allelic SNPs is performed to minimise erroneous calls which can look like tri and tetra allelic SNPs passing through filters NB The annotated SNP and indel matrices All indeis annotated txt and All SNPs annotated txt include tri allelic variants In addition ambiguous and non orthologous calls are flagged as and respectively Ortho SNP matrix nex includes SNP coordinates identified by GATK This file is directly importable into PAUP and is useful for phylogenetic estimations that require nucleotide data e g maximum likelihood Below is a screen capture of this file 11 nexus begin data dimensions ntax 17 nchar 106568 format symbols AGCT gap dat atype nucleotide transpose taxlabels REL606 M300 M331 M379 M615 M617 M618 M619 M620 M632 REL10938 REL1164A REL2179A REL4536A REL607 REL7177A REL8593A matrix NC_012967_58 GGGG NC_012967_64 T T T NC 012967 67 C C C NC 012967 171 NC 012967 392 NC 012967 437 NC 012967 464 NC 012967 473 NC 012967 479 NC 012967 509 NC 012967 536 NC 012967 558 NC 012967 587
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