Galaxy | Europe | Accessible Workflow | Pre-processing of scRNA-seq STRT-C1 Data

Pre-processing of scRNA-seq STRT-C1 Data

Step	Annotation
Step 1: Input dataset SRR1544693.fastq.gz select at runtime
Step 2: FastQC Short read data from your current history Output dataset 'output' from step 1 Contaminant list select at runtime Adapter list select at runtime Submodule and Limit specifing file select at runtime Disable grouping of bases for reads >50bp False Lower limit on the length of the sequence to be shown in the report Not available. length of Kmer to look for 7
Step 3: UMI-tools extract Library type Single-end Reads in FASTQ format Output dataset 'output' from step 1 Use Known Barcodes? No Method to extract barcodes Regular Expressions Barcode pattern for first read (?P<umi_1>.{6})(?P<discard_1>GGG){s<=1}(?P<cell_1>.{8}).* Is the barcode at the 5' end? True Output log? True Enable quality filter? No
Step 4: RNA STAR Single-end or paired-end reads Single-end RNA-Seq FASTQ/FASTA file Output dataset 'out' from step 3 Custom or built-in reference genome Use a built-in index Reference genome with or without an annotation use genome reference without builtin gene-model Select reference genome mm10full Gene model (gff3,gtf) file for splice junctions select at runtime Length of the genomic sequence around annotated junctions 100 Use 2-pass mapping for more sensitive novel splice junction discovery No None Empty. None Empty. Per gene/transcript output No per gene or transcript output Report chimeric alignments? Don't report chimeric alignments BAM output format specification: Read alignment tags to include in the BAM output NH (number of reported alignments/hits for the read) HI (query hit index) AS (local alignment score) nM (number of mismatches per (paired) alignment) ch (used to indicate chimeric alignments) HI tag values should be one-based None OneBestScore MAPQ value for unique mappers 60 Output filter criteria: Exclude the following records from the BAM output Nothing selected. Would you like to set additional output filters? No Algorithmic settings: Configure seed, alignment and limits options Use Defaults Performance tweaks / Troubleshooting: Number of genome bins for coordinate-sorting 50
Step 5: MultiQC Results Results 1 Which tool was used generate logs? STAR STAR outputs STAR output 1 Type of STAR output? Log STAR log output Output dataset 'output_log' from step 4 Report title Empty. Custom comment Empty. Use only flat plots (non-interactive images) False Output the multiQC log file? False
Step 6: Filter BAM dataset(s) to filter Output dataset 'mapped_reads' from step 4 Conditions Condition 1 Filters Filter 1 Select BAM property to filter on alignmentFlag Filter on this alignment flag 0 Condition 2 Filters Filter 1 Select BAM property to filter on alignmentFlag Filter on this alignment flag 16 Condition 3 Filters Filter 1 Select BAM property to filter on tag Filter on a particular tag nM:<3 Condition 4 Filters Filter 1 Select BAM property to filter on tag Filter on a particular tag NH:<2 Would you like to set rules? True Enter rules here (1 \| 2) & 3 & 4
Step 7: featureCounts Alignment file Output dataset 'out_file1' from step 6 Specify strand information Stranded (Forward) Gene annotation file featureCounts built-in Select built-in genome mm10 Output format Gene-ID "\t" read-count (MultiQC/DESeq2/edgeR/limma-voom compatible) Create gene-length file False Options for paired-end reads: Count fragments instead of reads Disabled; all reads/mates will be counted individually Only allow fragments with both reads aligned False Exclude chimeric fragments True Advanced options: GFF feature type filter exon GFF gene identifier gene_id On feature level False Allow reads to map to multiple features Disabled; reads that align to multiple features or overlapping features are excluded Minimum mapping quality per read 0 Exon-exon junctions False Long reads False Count reads by read group False Largest overlap False Minimum bases of overlap 1 Minimum fraction (of read) overlapping a feature 0 Minimum fraction (of feature) overlapping a read 0 Read 5' extension 0 Read 3' extension 0 Reduce read to single position Leave the read as it is Only count primary alignments False Ignore reads marked as duplicate False Annotates the alignment file with 'XS:Z:'-tags to described per read or read-pair the corresponding assigned feature(s). True Ignore unspliced alignments False
Step 8: UMI-tools count Sorted BAM file Output dataset 'output_bam' from step 7 Bam is paired-end False Umi Extract Method Barcodes are contained at the end of the read seperated by a delimiter Delimiter between read id and the UMI _ Method to identify group of reads Unique Edit distance threshold 1 Output a matrix of genes and cells, instead of a flat file True Extra parameters: Minimum mapping quality 0 Deduplicate per gene. XT Skip any reads where the gene matches this tag Empty. Deduplicate per contig (field 3 in BAM; RNAME) False Group reads only if they have the same cell barcode. True Random Seed 0 Prepend a label to all column headers No modifications

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