Trans-ABySS 1.4.4 User Manual
Contents
1. 1 reads_1_export fq sorted bam 1 reads_1_export fq sorted bam bai sim0003 contigs bam gt 1 reads_1_export fq sorted bam sim0003 contigs bam bai gt 1 reads_1_export fq sorted bam bai contigs fa are the various index files for the merged assembly sai are the suffix array index files sorted bam is the BAM file for each pair of read files bai are the BAM indexes When there is only one sorted bam contigs bam is a sym link to that sorted bam Otherwise contigs bam is the merged BAM file of all sorted bam C Align merged assembly to reference genome The merged assembly is split into multiple FASTA files where each contains at most 5000 contigs by default Then each FASTA file is aligned to the reference genome Transcriptome libraries e TA uses GMAP to align the merged assembly to the reference genome Genome libraries e TA uses BWA SW to align the merged assembly to the reference genome Example output contigs_to_genome sim0003 contigs cluster l J oa input l seq 1 fa output seq 1 sam seq fa are the split up FASTA files seq sam are the alignment output files The output files are in SAM format but TA also accepts alignments in PSL format such as those from BLAT k Create UCSC custom track of the merged assembly This stage is only applicable to transcriptome libraries Example output tracks cluster l e nan sim00032. mainly for debug purpose Format TO CO CC 11 12 AF1 AF2 where TO target overlap fraction overlap target_region1 target_region2 total_target_region_length CO contig overlap fraction overlap query_region1 query_region2 total_query_region_length CC contig coverage match_length1 match_length2 overlap query length I1 percent identity of alignment 1 I2 percent identity of alignment 2 AF1 alignment fraction of alignment 1 match_length1 query_length AF2 alignment fraction of alignment 2 can be sense fusion if the breakpoints reside in 2 transcripts and the orientations of the contig relative to the 2 transcripts are the same antisense fusion if the breakpoints reside in 2 transcripts and the orientations of the contig relative to the 2 transcripts are NOT the same LSR any fusion event not of the above types dbSNP entries for deletion events that are already annotated in dbSNP DGV entries for deletion and inversion events that are already annotated in DGV SNV INDEL snv_caller py Output Description events tsv events _filtered tsv events _filtered_novel tsv events_exons tsv events_exons_novel tsv LOG unfiltered snv indel events captured by gapped contig alignments filtered events lt event_reads gt gt min_reads_ contigs default 3 filtered events not annotated in dbSNP filtered non synonymous events residing in gene ex
3. command run and parameters used Contents of events filtered tsv Description Column Name 1 id 2 type 3 contig 4 transcript 5 gene 6 exons 7 align_blocks 8 geome_coord 9 contig_coord 10 splice event ID Each line represents an event captured by an individual contig Identical events will be linked by the first number of lt id gt Example 2 1 2 2 2 3 represent the same event captured by 3 different contigs Events are grouped by event type lt type gt and genome coordinate lt genome_coord gt event type Can be AS3 novel 3 splice site AS5 novel 5 splice site AS53 novel 5 and 3 splice site on the same alignment block novel_exon novel exon novel_intron novel intron novel_transcript novel transcript when contig cannot be mapped to any known transcript novel_utr novel UTR when novel alignment blocks exist beyond annotated 5 and 3 exons of mapped transcript retained_intron retained intron skipped_exon skipped exon contig ID transcript name gene name exon number s relative to transcript strand start from 1 alignment block numbers counted in ascending order of coordinate start from 1 genome coordinate of novel block Format chromosome start end contig coordinate of novel block splice sites adjacent to the novel junction E g gtAG U2 U12 11 12 13 14 15 multi_3 size orf Spanning_reads coverage where AG in capitals is
4. job id Note that predecessors_list_delimiter from jobs script cfg would be used here MEM is the amount memory to request for the job QUEUE is the list of cluster queues for the job THREADS is the number of CPUs for the parallel job FIRST_TASK_ID is the first task id for the array job LAST_TASK_ID is the last task id for the array job TMPMEM is the amount of disk space to request for the job SETUP_PATHS would be replaced with the command source path to setup CONTENT is the commands to be run in the job This variable is mandatory for all templates The following variables must be defined properly TMPDIR is the prefix for temporary files Typically the scheduler of your HPC cluster should configure it automatically for each job Otherwise please configure it in the template to use the cluster node s local temporary directory along with a unique prefix ie TMPDIR tmp JOB_ID TASK_ID QUEUE TA_JOBID is the task id of the array job This variable is mandatory for all array jobs in TA You should link this variable with the task id of the job ie TA_JOBID SGE_TASK_ID input An input file defines the set of libraries to process with TA There are no restrictions on the name and location of an input file This is the format of an input file LIBRARY ASSEMBLY_DIR PROJECT READLENGTH LIBRARYTYPE METALIBRARY e LIBRARY is the name of the library e ASSEMBLY_DIR is the path to the directory containin
5. merged best unique m90 gmap psl gz psl gz is the track that can be uploaded to the UCSC genome browser f Call fusion events and other large scale rearrangement events Example output fusions cluster ee Ried J LOG l l fusions tsv l yee A fusions tsv fusions_filtered tsv local tsv LOG See the next section for the description and format of output files i Call indels Example output indels cluster i fesse T LOG l l events tsv l J oa events tsv events_concat tsv events_exons tsv events_exons_novel tsv events_filtered tsv events_filtered_novel tsv filter_debug tsv LOG See the next section for the description and format of output files X Call novel splicing events and calculate coverage of known isoforms This stage is only applicable to transcriptome libraries Example output splicing cluster 1 LOG l coverage tsv l events tsv l log txt l mapping tsv events tsv events_filtered tsv l Sas coverage tsv events_summary tsv mapping tsv See the next section for the description and format of output files Output format of analyses results files Fusion fusion py Output Description fusions tsv unfiltered fusion events captured by split contig alignments fusions filtered tsv filtered events where num_read
6. the novel splice donor acceptor created by the novel sequence gt is the partner splice donor acceptor U2 U12 is name of the splice motif If a novel block creates two novel splice sites e g a skipped_exon event 2 splice sites will be reported e g gtAG U2 U12 GTag U2 U12 only applicable to retained_intron events True if the size of the intron retained is a multiple of 3 i e retained open reading frame size of novel block Only applicable to AS53 novel_exon novel_intron novel_transcript and novel_utr effect on open reading frame See below number of reads spanning novel junction gathered from reads to contig alignments number of reads spanning novel block Applies to AS53 novel_exon novel_transcript novel_utr and retained_intron Contents of coverage tsv Column Name Description 1 feature gene or transcript 2 model single letter initial of gene model used for coverage calculation The initial is specified in the configuration file model_matcher cfg 3 transcript transcript name gene gene name 5 exon exon number currently not relevant as exon level coverage is not reported strand transcript strand 7 coord coordinate of lt feature gt chromosome start end 10 11 12 13 feature size Best contig mapped to lt transcript gt in terms of bases covered bases_ reconstructed number of exonic bases reconstructed by all contig reconstructio
7. TA processes data on a per library basis Each library must belong to only one project but each project is expected to have multiple libraries In transcriptome cfg a project should be set up a new section Each project must have a working directory and a reference genome which are specified in topdir and reference respectively For example your_project_name topdir your transabyss working directory for this project reference name of the reference genome configured in genomes section abyss rmdups iterative cmd n LIB i INPUT_DIR o OUTPUT_DIR INDEL_ONLY t 12 abyss rmdups iterative mem 3G 12 bwa_sam tmpmem 60G samtofastq jar java java XX UseGCOverheadLimit Xmx10g You may override the defaults for processing each project with the postfixes cmd mem tmpmem java for the sections for command memory tmpmem and java respectively As shown here abyss rmdups iterative was configured use 12 threads and run on 12 CPUs and allocate 3G for each CPU to a total of 36G available memory configs genome cfg This configuration file serves the same purpose as transcriptome cfg except it is used for the genome pipelines configs model_matcher cfg This configuration file specifies the gene model files that are used by the module model_matcher py for contig transcript mapping Content of model_matcher cfg hg19 k knownGene_ref txt e ensGene_ref txt r refGene txt a acembly_ref txt order k e r a You should
8. Trans ABySS 1 4 4 User Manual Last updated October 10 2012 Written by Readman Chiu lt rchiu bcgsc ca gt Ka Ming Nip lt kmnip bcgsc ca gt Canada s Michael Smith Genome Sciences Centre BC Cancer Agency Vancouver BC Canada V5Z 4S6 Please direct your questions suggestions bug reports and feature requests to our Google Group at lt trans abyss googlegroups com gt Generating Assemblies with ABySS The input to Trans ABySS is one or more ABySS assemblies ABySS can be compiled as described in the README for ABySS http www bcgsc ca downloads abyss doc Should you run into any difficulties in compiling or running ABySS please contact the ABySS Google Group at lt abyss users googlegroups com gt Trans ABySS has been expanded to support 4 types of libraries each of which has its own assembly protocol 1 Transcriptome i assemble contigs at multiple k mer values with reads 2 Genome i assemble the unitigs at 2 k mer values with reads ii assemble the unitigs at a higher k mer value than those from i with reads and unitigs from i iii assemble contigs at the same k mer value from ii with reads and unitigs from ii Alternatively you may simply create one paired end assembly using only one k mer value Although simpler you may risk losing contigs for some genomic events 3 Targetted Genome i align reads to reference genome ii assemble contigs at multiple k mer values with reads aligned to region s of inter
9. _pairs gt min_read_pairs default 4 AND lt max_read_pairs default 2000 lt num_read_pairs gt lt flanking_pairs gt minimum lt breakpoint_pairs gt lt spanning_reads gt gt min_span_reads default 2 local tsv local events when alignment target regions overlap or alignment target regions overlap same gene or transcripts mapped by target regions overlap LOG run log recording command run and parameters used Content of fusion_filtered tsv Column Name Description 1 id event ID Each line represents an event captured by an individual contig Identical events will be linked by the first number of of id Example 2 1 2 2 2 3 represent the same event captured by 3 different contigs Events are grouped by type of rearrangement lt rearrangement gt and breakpoint lt breakpoint gt Reciprocal lt reciprocal gt events are indicated by a and b attached at the end Example 89a and 89b are reciprocal events contig contig ID contig size size or length of contig lt contig gt genomic_regions the 2 genomic regions the contig aligns to Format chromosomeA startl end1 chromosomeB start2 end2 chromosome names are the same as in the FASTA file used for contig alignments Order of regions is sorted by the chromosome names 5 contig_regions the corresponding contig coordinates of the 2 genomic regions Format startl end1 start2 end2 regions in the same order of
10. am of insertion If lt type gt del or inv lt ctg_start gt first base of deletion or deletion If lt type gt snv lt ctg_start gt lt ctg_end gt the base of substitution contig end coordinate If lt type gt ins or snv 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 len ref alt event_reads contig_reads genome_reads gene repeat length ctg_strand from_end confirm_contig_region within_simple_repeats repeatmasker within_segdup at_least_1_read_opposite dbsnp lt ctg_end gt lt ctg_end gt lt ctg_start gt If lt type gt del or inv last base of deletion or inversion length or size of event reference allele If lt type gt ins lt ref gt na alternative allele If lt type gt del lt ref gt na total number of reads spanning event from reads to contig alignment number of reads spanning event in contig lt ctg gt from reads to contig alignment total number of reads spanning event from reads to genome alignment gene in affected locus Format gene transcript intron exon number effect on open reading frame see below If the event size is bigger than 1 the output is a pairing of the above format on both coordinates i e geneA transcriptA intron exon numberA geneB transcriptB intron exon numberBleffect on open reading frame where A and B may be the same small event within t
11. cked for the fusion event indicates the contig aligns in the same direction of the gene strand indicates the contig aligns in opposite direction of the gene strand exon intron number exon intron N 5utr or 3utr where the breakpoints lie whether breakpoint is within exon boundaries yes or not no breakpoint coordinate lt breakpoint gt of reciprocal event captured in same library conventional nomenclature of rearrangement e g t 11 17 q12 2 q25 1 indicates which parts of the chromosomes are joined together L chromosome upstream of breakpoint coordinate R chromosome downstream of breakpoint coordinate gene name of the 5 transcript in sense_ fusion cases where the 5 and 3 transcripts can be unambiguously discerned otherwise gene name of the 3 transcript in sense_ fusion cases where the 5 and 3 transcripts can be unambiguously discerned otherwise exon number of the 5 gene where the breakpoint lies If breakpoint lies in an intron the downstream exon number will be reported If breakpoint lies in an UTR 5utr or 3utr will be 24 25 26 27 28 29 3 exon frame alignment_params type dbsnp dgv indicated exon number of the 3 gene where the breakpoint lies If breakpoint lies in an intron the downstream exon number will be reported If breakpoint lies in an UTR 5utr or 3utr will be indicated alignment details
12. coordinate of lt transcript gt are the same in annotation file 9 intronic intron number if lt contig gt is mapped to introns if otherwise 10 num_align_blocks number of alignment blocks 11 num_exons number of exons of lt transcript gt 12 num_matched_blocks number of alignment blocks matched to exons Internal blocks are considered matched when both edges align terminal blocks are considered matched when internal edges align 13 matched_blocks list of matched alignment blocks Blocks are numbered from left to right 14 matched_exons list of matched exons Exons are numbered in reference to transcript strand lt strand gt T5 score number of edges matched terminal edges count if corresponding internal edges match 16 coverage fraction of exonic bases of lt trancript gt covered reconstructed by lt contig gt 17 align_blocks genome coordinates start end of all alignment blocks with each block separated by Miscellaneous Open Reading Frame Effect Descriptors Throughout the output from TA a standard nomenclature used for example by the Human Genome Variation Society is used to denote the effect of an event on a gene at the protein level The following table describes the changes with an example notation and explanation Change Example frameshift A245Sfs Alanine 235 becomes Serine followed by a frameshift deletion V422 S431del deletion from Valine 433 to Serine 431 insertion Q484 485i
13. enome folder for quick random access to the reference sequence A lt genome gt 2bit file can be generated from the utility faToTwoBit available from http users soe ucsc edu kent src Running Trans ABySS All stages in TA are initiated with the Python driver script trans abyss py To show all available options in trans abyss py run this command python trans abyss py h Typically each stage can be run like so python trans abyss py lt stage gt p lt project gt l lt library gt a lt assembly dir gt L lt read length gt lt sample type gt Alternatively an input file can be used python trans abyss py lt stage gt n lt input file gt See Figure 1 for the workflow of the 10 stages in TA d setup directories 0 filter extend merge assemblies R prepare reads files PERSIE b align reads to reference genome r align reads to merged assembly c align merged assembly to reference genome f call fusions and large scale rearrangements fusion 4 7 aae xX call novel splicing and calculate coverage splicing k create UCSC custom track Figure 1 The workflow of Trans ABySS 1 4 4 Stages having the same color in this figure can be done in parallel d Set up directories TA sets up the output directories and makes sym links to your input ABySS assemblies from the assembly directory specified with a or within your input file Example output assembly in k62 gt ABySS Sam
14. est This is particularly useful when a subset of your dataset is interesting because the runtime is relatively short compared to assembling the whole genome 4 Strand Specific Transcriptome i align reads to reference genome ii divide the reads into batches for a plus strand fragments b minus strand fragments c unknown strand fragments according to the orientation of alignments from i iii assemble 2 sets of contigs at multiple k mer values one set using reads from batches ii a and ii c and another set using reads from batches ii b and ii c Currently TA provides limited support for this protocol For example TA only supports input reads in BAM files and the read aligner is limited to BWA only However you may also run the regular transcriptome pipeline on your strand specific transcriptome libraries We are currently working on a more sophisticated protocol for strand specific transcriptome libraries Installing Trans ABySS The TA package consists of the following files and directories bin setup configs input annotations utilities analysis sample_dataset bin TA requires the following external software packages pofware 1 3 2 Assembler for multiple kmer assemblies FEM Aligner of reads to merged assembly for genome libraries abyss map Aligner of reads to merged assembly for transcriptome libraries Aligner of a to reference genome for genome libraries owasw GMAP 2012 07 20 Aligner o
15. f contigs to reference genome for transcriptome or later libraries Pysam_ _ 0 6 Python interface for SAMtools It is recommended to put or sym link the executables of the above software in TA s bin directory Alternatively you may specify the paths in the setup file setup The setup file defines all environment variables required by TA Typically this command is included in nearly all job scripts created by TA source path to setup sh Content of setup export TRANSABYSS_VERSION 1 4 4 export TRANSABYSS_PATH trans abyss path export PYTHONPATH python path TRANSABYSS_PATH PYTHONPATH export ABYSSPATH abyss path export PICARD_DIR picard path export PATH TRANSABYSS_PATH bin java path ABYSSPATH PYTHONPATH PATH Please configure the setup file by giving each environment variable the correct path s configs configs transcriptome cfg This file contains the majority of the configurations for the transcriptome pipelines in TA It has the following major sections e commands This section contains the default commands for running each module e memory This section contains the default memory request for cluster jobs e genomes This section contains the paths to your reference genomes e tmpmem This section contains the default space request for temporary directories used in cluster jobs e java This section contains the java options used for each java package
16. g the library s multiple kmer assemblies e PROJECT is the project name of the library e READLENGTH is the smallest read length for the library e LIBRARYTYPE is the type of the library ie transcriptome genome targetted_genome plus strand minus_strand e METALIBRARY is the name for strand specific transcriptome library Not all fields are required If either option T or G or tG is used LIBRARYTYPE is not required ie LIBRARY ASSEMBLY_DIR PROJECT READLENGTH Otherwise LIBRARYTYPE is required ie LIBRARY ASSEMBLY_DIR PROJECT READLENGTH transcriptome LIBRARY ASSEMBLY_DIR PROJECT READLENGTH genome LIBRARY ASSEMBLY_DIR PROJECT READLENGTH targetted_genome Each strand specific transcriptome library consists of 2 lines one line for the plus strand and another line for the minus strand For example LIBOO1_plus assembly dir plus MyProject 100 plus_strand LIB001 LIBOO1_minus assembly dir minus MyProject 100 minus_strand LIBOO1 annotations Analysis modules of TA require comparisons to a reference genome and gene annotation files TA organizes annotation files by genome under the annotations folder for example annotations hg19 l genome 2bit l splice_motifs fa shared splice_motifs txt TA mainly uses the annotation files available from the UCSC genome browser ftp hgdownload cse ucsc edu goldenPath lt genome gt database A list of files required lt genome gt _annot txt and a dow
17. genomic regions 6 strands relative orientation of the 2 alignments in relation to the genome Format 7 flanking_pairs number of read pairs from reads to genome alignments with both mates flanking the breakpoint both pointing towards each other 10 11 12 13 14 15 16 17 18 19 20 21 22 23 breakpoint_pairs spanning_reads rearrangement breakpoint size genes transcripts senses exons introns exon_bounds reciprocal descriptor orientations 5 gene 3 gene 5 exon number of read pairs from reads to genome alignments with one mate spanning the breakpoint and the other mate flanking it both pointing towards each other This is useful for read support when reads lengths are long compared to fragment size Pairs up and down stream of the breakpoint are reported in a 2 member tuple number of reads spanning junction from reads to contigs alignments underlying genome rearrangement deduced by relative contig alignment orientations Can be translocation deletion inversion or duplication junction breakpoint Format chrA coordinate1 chrB coordinate2 Chromosome names are in same format as in FASTA file used for contig alignments size bp of the event genel gene2 of the genes involved in the fusion genel correspond to the first coordinate in breakpoint genel the second transcript1 transcript2 of the transcripts pi
18. he same transcript or different bigger events length of repeat in alternative allele e g AAAA 4 CAGCAG 2 query strand of alignment in relation to reference shortest distance bases of event to end of contig contig coordinate range start end used for checking event support in reads to contig alignments overlap with simple repeats Name of tandem repeat reported if overlap is True e g TRF_SimpleTandemRepeat_CATC if overlap is False overlap with RepeatMasker annotations Type of repeat reported if overlap is True e g AluSx LTR47A if overlap is False overlap with segmental duplication Segdup Chromosome Start_coordinate of segdup partner reported if overlap is True e g chr1 17048246 if overlap is False if at least 1 supporting read is aligned in opposite orientation to rest of supporting reads Can be true or false dbSNP entries if event is already annotated in dbSNP e g r 12028735 rs71510514 Novel Splicing model_matcher py Output Description events tsv events _filtered tsv events _summary tsv coverage tsv mapping tsv log txt LOG unfiltered novel splicing events not observed in annotations specified in model_matcher cfg filtered events See below for filtering criteria tally of filtered events by lt type gt transcript coverage mapping of contig to annotated transcripts detailed block by block mapping of alignments to exons run log recording
19. n num_reads bases reads depth mapped to lt feature gt fraction of exonic bases reconstructed by all contig mapped to lt feature gt number of reads spanning feature from reads to contigs alignments currently not reported total number bases spanning feature from reads to contigs alignments currently not reported lt bases_reads gt lt num_reads gt 14 15 16 17 18 19 contigs num_contigs best_contig align_blocks exons Contents of mapping tsv best_contig_reconstruction list of contigs mapped to lt feature gt number of contigs mapped to lt feature gt ID of contig that reconstructs lt feature gt best fraction of lt feature gt reconstructed by lt best_contig gt list of alignment blocks used for reconstructing lt feature gt only reported in intermediate batch outputs before filtering stage list of exons reconstructed only reported in intermediate batch outputs before filtering stage Colum Name Description n 1 contig contig ID 2 contig_len length or size of lt contig gt 3 coord genome alignment coordinate of lt contig gt 4 model single letter initial of gene model used for coverage calculation The initial is specified in the configuration file model_matcher cfg 5 transcript name of mapped transcript 6 gene gene name of lt transcript gt 7 strand strand of lt transcript gt 8 coding CODING or NONCODING NONCODING if start and end
20. nloading script lt genome gt _annot sh available for the genomes hg18 hg19 and mm9 are provided in the annotations folder for executing the wget downloads and running the following processing steps This is an example usage of setting up the hg19 annotation files cd lt TA_DIR gt annotations hgi9_annot sh hg19 hg19_annot txt hg19 lt TA_DIR gt where hg19 is the destination folder hg19 is the name of the genome Note that a snpixx txt gz is included in all genome s file lists This dbSNP file is used to annotate the snv indel events detected To speed up this annotation process the dbSNP annotation should be split by chromosome with this command split_dbsnp sh split_dbsnp sh lt TA_DIR gt annotations lt genome gt snp1ixx txt lt TA_DIR gt Note that dgv txt gz is also included This is the DGV database flat file used to annotate fusions and large scale rearrangement events detected The user is expected to have the single reference genome sequence FASTA file available on the cluster for contig alignments For example the reference genome hg19 can be downloaded from ftp ftp ncbi nih gov genbank genomes Eukaryotes vertebrates_mammals Homo_sapi ens GRCh37 special_requests After that put the path to the downloaded reference FASTA file in configs transcriptome cfg under genomes ie genomes hg19 path to your hg19 fasta_file here A 2bit version of the same genome sequence is expected to be present in the g
21. nsVA insertion of Valine and Alanine in between Glutamine 484 and Isoleucine 485 indel 293 Y294insKS Serine 293 to Tyrosine 294 becomes Lysine and Serine synon Synonymous silent substitution T327S Threonine 327 to Serine
22. ons filtered non synonymous events residing in gene exons not annotated in dbSNP run log recording command run and parameters used Content of events _filtered tsv Column Name Description 1 id type chr chr_start chr_end ctg ctg_len ctg_start ctg_end event ID Each line represents an event captured by an individual contig Identical events will be linked by the first number of of id Example 2 1 2 2 2 3 represent the same event captured by 3 different contigs Events are grouped by event type lt type gt coordinate lt chr gt lt chr_start gt lt chr_end gt and the alternative allele lt alt gt event type Can be snv ins del inv chromosome name as in the chromosome name in the FASTA file used for contig alignments chromosome start coordinate If lt type gt ins lt chr_start gt coordinate immediately upstream of insertion If lt type gt del or inv lt chr_start gt first base of deletion or inversion If lt type gt snv lt chr_start gt lt chr_end gt the base of substitution chromosome end coordinate If lt type gt ins or snv lt chr_end gt lt chr_start gt If lt type gt del or inv lt chr_end gt last base of deletion or inversion contig ID length of contig lt ctg gt that captures event contig start coordinate If lt type gt ins lt ctg_start gt coordinate immediately upstre
23. pleProject abyss 1 3 2 sim0003 k62 k74 gt ABySS SampleProject abyss 1 3 2 sim0003 k74 in is a text file listing all input read files it is an exact copy of the one in the assembly directory 0 Filter extend merge assemblies This stage is frequently referred to as FEM and it was part of Stage 0 in TA 1 3 Transcriptome libraries e junction contigs and indel bubbles are extended with abyss junction e short contigs and short islands are removed with abyss filtergraph Genome libraries e only indel bubbles are extended with abyss junction e no contigs are filtered by length If there is no reference genome for your library you may stop after this stage Example output filter cluster l yee a k62 l tee J k74 sim0003 b fa sim0003 contigs fa sim0003 f fa sim0003 j fa sim0003 nb path sim0003 74 abyss ta filter COMPLETE merge cluster sim0003 contigs fa sim0003 merge abyss rmdups iterative COMPLETE stats txt filter k b fa contains extended indel bubbles filter k f fa contains contigs passing the length filter filter k j fa contains extended junction contigs filter k contigs fa is the concatenate of b fa f fa and j fa merge contigs fa is the merged assembly merge stats txt contains the statistics for the ABySS assemblies and the merged assembly R Prepare reads If your read files are FASTQ files you may
24. set up one section for each reference genome you use in TA The gene model files referenced in each section are expected to be found in the annotations directory See annotations for instructions on downloading annotation files Each gene model file is assigned an alias for quick referencing For example e represents the Ensembl gene model file while r represents the Refseq gene model file These aliases should be arranged in a comma separated list in the order field from highest priority to lowest priority Priority set here will be used in breaking ties when a contig can be mapped to genes from multiple models configs job_script cfg This configuration file contains the configurations for job submissions Content of job_script cfg local gsc_local txt cluster_basic gsc_sge_basic txt cluster_parallel gsc_sge_parallel txt cluster_basic_array gsc_sge_basic_array txt cluster_parallel_array gsc_sge_parallel_array txt predecessors_list_delimiter run_local_job_command bash submit_cluster_job_command qsub submit_cluster_job_return_string Your job JOBID has been submitted submit_cluster_array_job_return_string Your job array JOBID has been submitted e local defines the template for local jobs e cluster_basic defines the template for basic single CPU cluster jobs e cluster_parallel defines the template for parallel multiple CPUs cluster jobs e cluster_basic_array defines the template for basic array clu
25. skip this stage Example output reads_to_contigs cluster reads l 1 reads_1_export fq 2 reads_2_export fq sim0003 in in is a text file listing all input read files b Align reads to reference genome TA does not do anything for this stage out of the box This stage is meant to be done on your own Transcriptome libraries e You must align reads to the genome and exon exon junction reference with JAGuaR or other gap aligner such as GSNAP Genome libraries e You may align reads to the reference genome with any short read aligner such as BWA that outputs in SAM format No matter which ever route you take you must create one indexed BAM file in the reads to genome directory This indexed BAM file is required in stage f for both transcriptome and genome libraries and in stage i for genome libraries only r Align reads to merged assembly Transcriptome libraries e TA is defaulted to use BWA to align reads to the merged assembly Genome libraries e TA is defaulted to use abyss map to align reads to merged assembly Example output reads_to_contigs cluster l S nan reads yee a sim0003 in sim0003 contigs fa gt merge sim0003 contigs fa sim0003 contigs fa fai sim0003 contigs fa amb sim0003 contigs fa ann sim0003 contigs fa bwt sim0003 contigs fa pac sim0003 contigs fa sa 1 reads_1_export fq sai 2 reads_2_export fq sai
26. ster jobs e cluster_parallel_array defines the template for parallel array cluster jobs e predecessors_list_delimiter defines the delimiter for the list of predecessors for each job e run_local_job_command defines the command to run local jobs e submit_cluster_job_command defines the command to submit batch jobs e submit_cluster_job_return_string defines the string returned when batch jobs are submitted This string is used for retrieving the job id from a batch job submitted with the submit_cluster_job_command JOBID corresponds to the part the string representing the job id You may use Python s regular expressions http docs python org library re html in this string submit_cluster_array_job_return_string defines the string returned when array jobs are submitted Its purpose is same as submit_cluster_job_return_string configs templates We attempt to use job script templates to simplify the process of setting up batch job submission of TA jobs in different HPC environment Although our templates were written to work with the Sun Grid Engine of our cluster you can create your own templates for your HPC environment The following variables in templates would be replaced with the appropriate values when job scripts are generated JOB_NAME is the name of the job WORKING_DIR is the working directory of the job The stdout and stderr logs would be place in this directory PREDECESSoRS is the list of predecessors
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