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SARS-CoV-2: map ONT reads to transcripts

Annotation: Identify viral reads from mapping against a combined viral/host reference, then realign the viral reads with settings optimized for aligning to "spliced" sgRNAs

Step	Annotation
Step 1: Input dataset ONT sequenced reads select at runtime
Step 2: Input dataset Combined host/viral reference select at runtime
Step 3: Input parameter Name of the SARS-CoV-2 genome Not available.
Step 4: NormalizeFasta FASTA dataset or dataset collection Output dataset 'output' from step 2 The line length to be used for the output fasta file 80 Truncate sequence names at first whitespace False
Step 5: Map with minimap2 Will you select a reference genome from your history or use a built-in index? Use a genome from history and build index Use the following dataset as the reference sequence Output dataset 'outFile' from step 4 Single or Paired-end reads Single Select fastq dataset Output dataset 'output' from step 1 Select a profile of preset options Long-read spliced alignment (-k15 -w5 --splice -g2000 -G200k -A1 -B2 -O2,32 -E1,0 -C9 -z200 -ub --splice-flank=yes). In the splice mode, 1) long deletions are taken as introns and represented as the `N' CIGAR operator 2) long insertions are disabled 3) deletion and insertion gap costs are different during chaining 4) the computation of the `ms` tag ignores introns to demote hits to pseudogenes. (splice) Indexing options: Use homopolymer-compressed k-mer ? False k-mer size 13 minimizer window size Not available. split index for every N input gigabases Not available. Mapping options: retain at most INT secondary alignments 32 Max fragment length for PE alignment Not available. filter out top FLOAT fraction of repetitive minimizers Not available. force minimap2 to always use k-mers occuring this many times or fewer Not available. stop chain enlongation if there are no minimizers in INT-bp Not available. bandwidth used in chaining and DP-based alignment Not available. minimal number of minimizers on a chain Not available. minimal chaining score (matching bases minus log gap penalty) Not available. Maximum seed skips during chaining Not available. Maximum number of partial chains checked during chaining Not available. skip self and dual mappings (for the all-vs-all mode) False min secondary-to-primary score ratio Not available. Alignment options: Customize spliced alignment mode? Yes, enable spliced alignments (--splice) Maximum allowed gap on the reference Not available. Cost of non-canonical (non-GT-AG) splicing Not available. how to find GT-AG don't match GT-AG (-un) Assume conserved flanking region of splice sites? True Use previously annotated splice sites to guide the alignment? No, perform unbiased alignment Score for a sequence match Not available. Penalty for a mismatch Not available. Gap open penalties for deletions Not available. Gap open penalties for insertions Not available. Gap extension penalties; a gap of size k cost '-O + -Ek'. If two numbers are specified, the first is the penalty of extending a deletion and the second for extending an insertion Not available. Gap extension penalty for extending an insertion; if left empty uses the value specified for Gap extension penalties above Not available. Z-drop threshold for truncating an alignment Not available. Z-drop threshold for reverse-complementing the query Not available. minimal peak DP alignment score Not available. Filter seeds towards the ends of chains before performing base-level alignment? True Set advanced output options:* Select an output format BAM don't output base quality False write CIGAR with >65535 ops to the CG tag False minibatch size for mapping (in megabyte) Not available. Output cs tag? Nothing selected. Generate CIGAR False write =/X CIGAR operators False use soft clipping for supplementary alignments ? False
Step 6: Filter FASTA FASTA sequences Output dataset 'outFile' from step 4 Criteria for filtering on the headers Regular expression on the headers Regular expression pattern the header should match Not available. Criteria for filtering on the sequences No filtering Remove duplicate sequences False Output discarded FASTA entries False
Step 7: Samtools view SAM/BAM/CRAM data set Output dataset 'alignment_output' from step 5 What would you like to look at? A filtered/subsampled selection of reads Configure filters: Filter by regions Manualy specify regions Filter by regions Not available. Filter by readgroup No Filter by quality Not available. Filter by library Empty. Filter by number of CIGAR bases consuming query sequence Not available. Require that these flags are set Nothing selected. Exclude reads with any of the following flags set Nothing selected. Exclude reads with all of the following flags set Nothing selected. Configure subsampling: Subsample alignment Specify a downsampling factor Downsampling factor 1.0 Seed for random number generator Not available. What would you like to have reported? All reads retained after filtering and subsampling Produce extra dataset with dropped reads? False Read Reformatting Options: Strip read tags from outputs Collapse backward CIGAR operation False Output format BAM (-b) None -b Reference data No, see help (-output-fmt-option no_ref)
Step 8: Samtools fastx BAM or SAM file to convert Output dataset 'outputsam' from step 7 Output format compressed FASTQ Default quality if none is given Not available. Output quality in the OQ tag if available False add Illumina Casava 1.8 format entry to header (eg 1:N:0:ATCACG) False outputs others Copy RG/BC/QT tags to output header False copy arbitrary tags to the FASTA header line Empty. Omit or append read numbers no Require that these flags are set Nothing selected. Exclude reads with any of the following flags set Nothing selected. Exclude reads with all of the following flags set Nothing selected. Write index read files No
Step 9: Map with minimap2 Will you select a reference genome from your history or use a built-in index? Use a genome from history and build index Use the following dataset as the reference sequence Output dataset 'output' from step 6 Single or Paired-end reads Single Select fastq dataset Output dataset 'output' from step 8 Select a profile of preset options Nothing selected. Indexing options: Use homopolymer-compressed k-mer ? False k-mer size 8 minimizer window size 1 split index for every N input gigabases Not available. Mapping options: retain at most INT secondary alignments 32 Max fragment length for PE alignment Not available. filter out top FLOAT fraction of repetitive minimizers Not available. force minimap2 to always use k-mers occuring this many times or fewer Not available. stop chain enlongation if there are no minimizers in INT-bp 30000 bandwidth used in chaining and DP-based alignment Not available. minimal number of minimizers on a chain Not available. minimal chaining score (matching bases minus log gap penalty) Not available. Maximum seed skips during chaining 40 Maximum number of partial chains checked during chaining Not available. skip self and dual mappings (for the all-vs-all mode) False min secondary-to-primary score ratio 0.7 Alignment options: Customize spliced alignment mode? Yes, enable spliced alignments (--splice) Maximum allowed gap on the reference 30000 Cost of non-canonical (non-GT-AG) splicing 0 how to find GT-AG don't match GT-AG (-un) Assume conserved flanking region of splice sites? False Use previously annotated splice sites to guide the alignment? No, perform unbiased alignment Score for a sequence match 1 Penalty for a mismatch 2 Gap open penalties for deletions 2 Gap open penalties for insertions 24 Gap extension penalties; a gap of size k cost '-O + -Ek'. If two numbers are specified, the first is the penalty of extending a deletion and the second for extending an insertion 1 Gap extension penalty for extending an insertion; if left empty uses the value specified for Gap extension penalties above 0 Z-drop threshold for truncating an alignment 400 Z-drop threshold for reverse-complementing the query 200 minimal peak DP alignment score Not available. Filter seeds towards the ends of chains before performing base-level alignment? False Set advanced output options:* Select an output format BAM don't output base quality False write CIGAR with >65535 ops to the CG tag False minibatch size for mapping (in megabyte) Not available. Output cs tag? Nothing selected. Generate CIGAR False write =/X CIGAR operators False use soft clipping for supplementary alignments ? False
Step 10: Samtools view SAM/BAM/CRAM data set Output dataset 'alignment_output' from step 9 What would you like to look at? A filtered/subsampled selection of reads Configure filters: Filter by regions No Filter by readgroup No Filter by quality 1 Filter by library Empty. Filter by number of CIGAR bases consuming query sequence Not available. Require that these flags are set Nothing selected. Exclude reads with any of the following flags set Alignment of the read is not primary Alignment is supplementary Exclude reads with all of the following flags set Nothing selected. Configure subsampling: Subsample alignment Specify a downsampling factor Downsampling factor 1.0 Seed for random number generator Not available. What would you like to have reported? All reads retained after filtering and subsampling Produce extra dataset with dropped reads? False Read Reformatting Options: Strip read tags from outputs Collapse backward CIGAR operation False Output format BAM (-b) None -b Reference data No, see help (-output-fmt-option no_ref)
Step 11: bamCoverage BAM/CRAM file Output dataset 'outputsam' from step 10 Bin size in bases 1 Scaling/Normalization method Do not normalize or scale Coverage file format bigwig Compute an exact scaling factor False Region of the genome to limit the operation to Empty. Show advanced options no

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wolfgang-maier

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