Galaxy | Europe | Published Workflow | STRT-Seq Workflow (with barcodes) (rename sequences)

STRT-Seq Workflow (with barcodes) (rename sequences)

Step

Annotation

Step 1: Input dataset

FASTQ Data

select at runtime

Step 2: Input dataset

Barcodes File

select at runtime

Step 3: Input dataset

mm10 GTF

select at runtime

Step 4: Rename sequences

Input file in FASTA or FASTQ format

Output dataset 'output' from step 1

Rename sequence identifiers to

Numeric Counter

Fix Weird Header Issue

Step 5: UMI-tools extract

Library type

Single-end

Reads in FASTQ format

Output dataset 'output' from step 4

Use Known Barcodes?

Yes

Barcode File

Output dataset 'output' from step 2

Apply correction to cell barcodes?

False

Method to extract barcodes

Regular Expressions

Barcode pattern for first read

(?P<umi_1>.{6})(?P<discard_1>GGG)(?P<cell_1>.{8}).*

Is the barcode at the 5' end?

True

Output log?

True

Enable quality filter?

Step 6: RNA STAR

Single-end or paired-end reads

Single-end

RNA-Seq FASTQ/FASTA file

Output dataset 'out' from step 5

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

Output dataset 'output' from step 3

Length of the genomic sequence around annotated junctions

100

Use 2-pass mapping for more sensitive novel splice junction discovery

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

Output filter criteria:

Exclude the following records from the BAM output

Nothing selected.

Would you like to set additional output filters?

Algorithmic settings:

Configure seed, alignment and limits options

Use Defaults

Performance tweaks / Troubleshooting:

Number of genome bins for coordinate-sorting

Step 7: UMI-tools deduplicate

Reads to deduplicate in SAM or BAM format

Output dataset 'mapped_reads' from step 6

extract_umi_method

Read ID

Separator between read id and UMI.

Tag which contains UMI.

Empty.

Method used to identify PCR duplicates within reads.

Reads group share the exact same UMI

Edit distance threshold

BAM is paired end

False

Spliced reads are unique

False

Soft clip threshold

Use the read length as as a criterion when deduping

False

Consider all alignments to a single contig together

False

Only consider a random selection of the reads

1.0

Only consider a single chromosome

False

Deduplicate per contig

False

Deduplicate per gene

False

Tabular file mapping genes to transripts

select at runtime

Deduplicate by this gene tag

Empty.

Step 8: featureCounts

Alignment file

Output dataset 'output' from step 7

Specify strand information

Unstranded

Gene annotation file

in your history

Gene annotation file

Output dataset 'output' from step 3

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

Exon-exon junctions

False

Long reads

False

Count reads by read group

False

Largest overlap

False

Minimum bases of overlap

Minimum fraction (of read) overlapping a feature

Minimum fraction (of feature) overlapping a read

Read 5' extension

Read 3' extension

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 9: UMI-tools count

Sorted BAM file

Output dataset 'output_bam' from step 8

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

Output a matrix of genes and cells, instead of a flat file

True

Extra parameters:

Minimum mapping quality

Deduplicate per gene.

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

Prepend a label to all column headers

No modifications

Step 10: summarise counts

Step 1: Input dataset input select at runtime
Step 2: Table Compute Input Single or Multiple Tables Single Table Table Output dataset 'output' from step 1 Input data has Column names on the first row Row names on the first column Advanced File Options : Header begins at line N Not available. Read N lines only Not available. Skip N lines from bottom Not available. Skip blank lines True Type of table operation Compute expression across rows or columns Calculate Sum For each Column Output formatting options Ignore NA values Output column headers Output row headers Output decimal precision 6
Step 3: Table Compute Input Single or Multiple Tables Single Table Table Output dataset 'table' from step 2 Input data has Column names on the first row Row names on the first column Advanced File Options : Header begins at line N Not available. Read N lines only Not available. Skip N lines from bottom Not available. Skip blank lines True Type of table operation Compute expression across rows or columns Calculate Custom Function Custom function on 'vec' vec.describe() For each Column Output formatting options Ignore NA values Output column headers Output row headers Output decimal precision 6

About this Workflow

Author

mehmet-tekman

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STRT-Seq Workflow (with barcodes) (rename sequences)

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