CASAVA Release Notes
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1 CASAVA Release Ntes CASAVA Release Ntes New Features: CASAVA is the first versin f the sftware package that supprts the Nextera dual indexing prtcl. Single index data cntinue t be supprted in CASAVA as befre. The applicatin f dual indexing in CASAVA is described belw: In the sample sheet, a dash sign ( ) shuld be used t separate the tw indexes in the Index clumn. In the demultiplexing wrkflw (cnfigurebcltfastq.pl), the use bases mask parameter will need t be included if dual indexing is indicated in the sample sheet: Example: use bases mask=y*,i*,i* (fr single end runs) r use bases mask=y*,i*,i*,y* (fr paired end runs) The parameter mismatches has als been mdified t supprt dual indexing. Example: mismatches=1,1 (allws 1 base mismatch in each f the index reads) By default, CASAVA will nt allw mismatches in either index read ( mismatches=0,0). CASAVA is als the first versin f CASAVA t perfrm adapter masking. The applicatin f adapter masking in CASAVA is described belw: In the demultiplexing wrkflw (cnfigurebcltfastq.pl), the adapter sequence parameter is used t indicate the lcatin f the FASTA file cntaining the adapter sequence(s) t be masked. If nly ne sequence is specified within the FASTA file, the same sequence is used fr all sequencing reads. If multiple sequences are specified, the first sequence is used fr the first sequencing read, the secnd sequence fr the secnd sequencing read, and s n. Index reads are nt affected by the adapter masking. Imprvements By default, fastq.gz files generated thrugh the demultiplexing wrkflw (BcltFastq.pl) nw nly cntain passfilter (PF) reads. T include all reads, the with failed reads parameter needs t be included in the demultiplexing wrkflw (cnfigurebcltfastq.pl). CASAVA nw includes the index sequence in the header f undetermined fastq files t help trublesht ptential indexing issues. CASAVA can nw generate ne fastq file per read if the parameter fastq cluster cunt is set t 0 in the demultiplexing wrkflw (cnfigurebcltfastq.pl). Page 1 f 7
2 CASAVA Release Ntes Minr bug fixes Issue that caused the Align as yu g wrkflw t fail has been addressed. Please see CASAVA Release Ntes fr the Align as yu g wrkflw. Archival BAM nw includes bth mapped and unmapped read pairs. QseqtFastq nw prperly designates PF/nn PF reads. 2 f 7
3 CASAVA Release Ntes CASAVA Release Ntes (pg. ##) User Guide page number The primary scripts fr CASAVA secndary analysis have changed in CASAVA The new required wrkflws are as fllws: (pg.22) cnfigurebcltfastq.pl Script which cnverts a directry f BCL files (frm RTA/OLB) int a directry f cmpressed Fastq files, separated by Prject/Sample. An example cmmand line is shwn belw: {CASAVA Install Directry}/bin/cnfigureBclTFastq.pl (pg. 36) CASAVA expects this script t be run in the BaseCalls flder by default. T specify anther input directry, the parameter input dir shuld be used. (pg. 36) The results will be placed int the flder BaseCalls/../../../Unaligned by default. T specify anther utput directry, the parameter utput dir shuld be used. (pg. 36) A sample sheet is required fr Indexed Runs r Runs where nly a subset f lanes will be analyzed. T specify the lcatin f a sample sheet, the parameter sample sheet shuld be used. The sample sheet is ptinal fr all ther Runs. (pg. 165) T cnvert a directry f qseq files, please use the cnfigureqseqtfastq.pl script. (pg. 36) By default, the number f clusters per cmpressed fastq file is set at 4,000,000. T adjust this number, use the parameter fastq cluster cunt (pg. 48) cnfigurealignment.pl (frmerly GERALD.pl) Script which cnfigures alignment n a directry f cmpressed Fastq files, and creates alignment directries separated by Prject/Sample. An example cmmand line is shwn belw: {CASAVA Install Directry}/bin/cnfigureAlignment.pl {Path t cnfig file} make NOTE: CASAVA expects this script t be run in the Unaligned flder by default. T specify anther input directry, the parameter EXPT_DIR shuld be used. NOTE: The results will be placed int the flder {Run Flder}/Aligned by default. T specify a different utput directry, the parameter OUT_DIR shuld be used. NOTE: By default, ELAND_FASTQ_FILES_PER_PROCESS is set at 3. If yu wish t adjust this value, please ensure that the fastq cluster cunt value (frm cnfigurebcltfastq.pl) multiplied by the ELAND_FASTQ_FILES_PER_PROCESS value equals between millin reads t ensure ptimal ELAND perfrmance. (pg. 88) cnfigurebuild.pl (frmerly run.pl) Script which cnfigures variant calling n ne r mre Sample Alignment directries. {CASAVA Install Directry}/bin/cnfigureBuild.pl id {Path t Sample Alignment Directry} and either refsequences={path t Single Entry Fasta Directry } r samtlsreffile={ Path t Multi Reference Fasta File} NOTE: The results will be placed int the flder {Run Flder}/Build by default. T specify a different utput directry, the parameter d shuld be used. Page 3 f 7
4 NOTE: The refsequences parameter shuld be used if ELAND_GENOME was used fr alignment. The samtlsreffile parameter shuld be used if SAMTOOLS_GENOME was used fr alignment. NOTE: T cnfigure RNA variant calling analysis, please use the script cnfigurernabuild.pl (frmerly runrna.pl) General Updates (pg. 116) CASAVA requires Cmake versin 2.8 and Bst versin Bth packages will be installed autmatically if nt detected in yur envirnment during installatin. Full paths shuld be used fr all cmmand line ptins fr all wrkflw scripts. BCL t Fastq Cnverter (cnfigurebcltfastq.pl) (pg. 39) The utput results fr Fastq cnversin and alignment will be rganized by prject. The prject flders allw users t naturally grup samples prcessed n a flw cell. Within the prjects flder, there will be a flder fr each sample. Within a sample flder, the FASTQ files fr the sample will be written such that each file is f a cnvenient size fr dwnstream prcessing. The name f each FASTQ file prvides the sample, the index used, the lane, and whether the file cntains the first r secnd read. The names are based n infrmatin btained frm the sample sheet. If n sample sheet is prvided, a default sample sheet is assumed. The same flder structure will accmmdate single sample and indexed runs. The Cnverter expects the fllwing RTA utputs (depending n versin): RTA 1.8 and previus CASAVA expects ps.txt files in the Intensities directry and filter files in the BaseCalls directry. RTA 1.9 CASAVA expects.lcs in the Intensities/L00* directries and filter/cntrl files in the BaseCalls/L00* directries. RTA CASAVA expects.clcs in the Intensities/L00* directries and filter/cntrl files in the BaseCalls/L00* directries. NOTE: All reads are included in the FASTQ files, nt just filtered reads. There is n ptin t parse ut the nnpassing filter reads frm the generated FASTQ files. Alignment (cnfigurealignment.pl) (pg. 57) The ELAND_GENOME parameter needs t be pinted t a flder f regular (nn squashed) fasta files. Yu can als use the SAMTOOLS_GENOME t pint t a single multi entry fasta file. (pg. 58) Semi repeat reslutin and the rphan aligner are new features in ELAND, and are turned n by default in CASAVA 1.8. T use full repeat reslutin (which gives increased alignment % but takes almst twice as lng t run as semi repeat reslutin), use the fllwing parameter: INCREASED_SENSITIVITY sensitive. 4 f 7
5 (pg. 53) If yu use the SAMTOOLS_GENOME parameter and yur fasta files cntain characters that CASAVA 1.8 restricts, yu can chse t ignre this restrictin t perfrm alignment by adding the fllwing parameter t the GERALD cnfig file: CHROM_NAME_VALIDATION ff NOTE: ELAND v2e (specifically the rphan aligner) cannt prcess chrmsme names with a cln (:) r backslash (\). These are restricted characters by default, but can be used if the CHROM_NAME_VALIDATION is turned ff. It is nt recmmended t use fasta files that cntain clns r backslashes in the header entries. (pg. 54) Fr eland_rna, the new parameter ELAND_RNA_GENOME_ANNOTATION can be pinted at either a refflat.txt.gz r seq.gene.md file. (pg. 67) The parameter DATASET_POST_RUN_COMMAND can be used in the cnfig file t specify cmmands t be run upn the cmpletin f alignment n each FASTQ files. T specify a cmmand t be run at the cmpletin f all analysis, please use the parameter POST_RUN_COMMAND n the make cmmand line, as shwn in the example belw: make j 8 POST_RUN_COMMAND:= ech Alignment cmpleted Pst-Alignment (cnfigurebuild.pl/cnfigurernabuild.pl) (pg. 103) Read alignments are nw stred in the pst alignment build as BAM files. These files can be fund in the Parsed directry as {chr. Name}/bam/srted.bam. The BAM utput fr RNA Seq builds nw represents each read which crsses a splice junctin as a single recrd, using the CIGAR 'N' character t indicate an alignment spanning an intrn. This shuld allw the alignments t be mre easily viewed in the Brad IGV and used in third party analysis tls. (pg. 90) Pst alignment can nw be run in archival mde. In this mde all reads prvided as input fr pstalignment will be stred in their entirety in the pst alignment BAM files and the entire build can be ptinally cnslidated t a single archival BAM file. This mde can be activated by using the " srtkeepallreads" flag. Nte the fllwing changes t the BAM files in archival mde: Purity filtered and PCR duplicate reads will be stred in the chrmsme BAM files with the apprpriate anntatins. These reads will nt be used fr variant calling r RNA Cunting. A new directry will be created in the Parsed flder called 'ntmapped'. This directry will cntain all read pairs where bth reads culd nt be mapped, r single reads which culd nt be mapped in a single ended build. These reads are divided int the fllwing categries: 1) whether n match was fund, 2) the reads culd nt be uniquely placed, 3) a qc failure ccurred, r 4) the reads were repeatmasked by ELAND. (pg. 147) CASAVA 1.8 uses a new snp caller which mdels the prbability distributin f diplid gentypes at each site. Snp and site gentype files nw prvide a mst likely gentype tgether with snp and gentype Q scres fr each site. 5 f 7
6 (pg. 141) The CASAVA 1.8 indel caller nw prvides calls fr dense, verlapping and larger indels. By default indels f up t 300 bases will be gentyped fr a paired end run. In additin the indel caller nw reprts and gentypes pen ended breakpints fr all ther large, cmplex r incmplete indels. New Summary Dcuments (pg. 42) Demultiplex_Stats.htm Summary File created at the end f the BCL t Fastq Cnverter (cnfigurebcltfastq.pl). Lcated in the Unaligned/Basecall_Stats_{Flw Cell ID} flder Includes a summary f Barcde Lane Statistics and Sample Infrmatin fr the entire Flw Cell (pg. 74) Sample_Summary.htm Prject Summary File created at the end f the prject Alignment (cnfigurealignment.pl) Lcated in the Aligned/{Prject Name}/ Summary_Stats_{Flw Cell ID} Barcde_Lane_Summary.htm mre detailed summary file, per barcde lane. Lcated in the Aligned/{Prject Name}/ Summary_Stats_{Flw Cell ID} Mismatch Rate is the new term fr Errr Rate, t accunt fr the fact that mismatches include sequencing errrs as well as SNV s. (pg. 79) Flwcell_Summary_{Flw Cell ID}.htm Summary File created at the end f the Alignment (cnfigurealignment.pl) Lcated in the Aligned flder A summary f all Sample_Summary files (fr all Prjects n the Flw Cell). Align-as-yu-g (pg. 38) CASAVA 1.8 prvides a mechanism t align the first read f a paired end run befre cmpletin f the run. This can be dne by using the target r1 available in the Makefiles fr bth the BCL t FASTQ cnverter and the alignment. These targets can be invked at any time after the last read has started (after cmpletin f the indexing read fr multiplexed runs). They are invked by typing the fllwing cmmand while in the apprpriate directry: make r1 As usual, the " j <n>" cmmand line ptin is supprted t indicate up t <n> prcesses in parallel. Hwever, fr the BCL cnversin, the maximum level f parallelizatin is 8. Fr instance, assuming that the BCL cnversin t FASTQ and the alignment have been cnfigured at the default lcatins (Unaligned and Aligned subdirectries f the run flder) and that the current directry is the run flder, the cnversin and the alignment f the first read can be dne using the fllwing cmmands: 6 f 7
7 cd Unaligned make j 8 r1 cd../aligned make j 16 r1 Fr the situatin where the jbs are submitted t a queuing system, r any situatin where the cnversin f the BCLs has t be asynchrnus, it is pssible t use the Makefile variable POST_RUN_COMMAND_R1 t autmatically start the alignment f read 1 at the end f the BCL cnversin. That variable can be set either as an envirnment variable, r n the make cmmand line, in which case the cmmand fr bth the cnversin and the alignment wuld be: cd Unaligned make j 8 r1 POST_RUN_COMMAND_R1="cd../Aligned ; make j 16 r1" Validatin Datasets Tw validatin datasets have been included with CASAVA specifically t ensure yur versin f CASAVA has been successfully installed. These datasets start with BCL files and run all the way thrugh Variant Calling. After the cmpletin f each validatin run, CASAVA will print ut whether each main part f CASAVA Demux (BCLtFastq), Alignment, and Build (Variant Calling) has Passed r Failed. NOTE: These datasets d nt represent the quality f actual sequencing data generated using Illumina technlgy. The results frm the validatin datasets were nt designed t be analyzed further. T run end t end validatin n the DNA (small) test data set included with CASAVA, yu can use the fllwing cmmands: {CASAVA Install Directry}/bin/cnfigureValidatin.pl utput dir./validatindefault make C ValidatinDefault use make j <parallel jbs> t speed up the cnversin T run end t end validatin n the indexed RNA (large) test data set included with CASAVA, yu can use the fllwing cmmands: {CASAVA Install Directry}/bin/cnfigureValidatin.pl surce dir {CASAVA Install Directry}/share/CASAVA 1.8.0/examples/Validatin/110120_P20_0993_A805CKABXX utput dir./validatinmultiplexed make C ValidatinMultiplexed use make j <parallel jbs> t speed up the cnversin
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