Flags

Required flags

Datastructure flags

Flag

Input type

Description

--db

PATH

The directory containing the preindexed Kraken or KrakenUniq database (see: quicksand_build-page):

Input:

refseq
  ├── kraken
  │    └── Mito_db_kmer22
  │           ├── taxonomy
  │           ├── ...
  │           └── database.kdb


Example:

--db Path/to/refseq/kraken/Mito_db_kmer22

--genomes

PATH
The directory containing the indexed FASTA-FILES of the reference genomes that were used to build
the kraken database. Format PATH/${family}/${species}.fasta (see: quicksand_build-page)
Input:

refseq
  ├── genomes
  │    └── Hominidae
  │           ├── Homo_sapiens.fasta
  │           └── ...


Example:

--genomes Path/to/refseq/genomes

--bedfiles

PATH
The directory containing the dustmasked BED-FILES of the reference genomes FASTA-FILES
Format DIR/${species}_masked.bed (see: quicksand_build-page)
Input:

refseq
  ├── masked
  │      ├── Homo_sapiens_masked.bed
  │      └── ...


Example:

--bedfiles Path/to/refseq/masked
Input flags

quicksand contains an optional demultiplexing preprocessing. However, it is an inhouse demultiplexer working only on bam-files provided by the MPI EVA Core Unit. For the processing of data coming from the MPI EVA run quicksand with the --bam PATH and --rg PATH flags as alternative to the --split PATH parameter.

Flag

Input type

Description

--split

PATH
Standard input
A directory containing the demultiplexed, adapter trimmed (and overlap-merged) input files
Input:

split
  ├── RG1.bam
  ├── RG2.fastq
  ├── RG3.fastq.gz
  ├── RG4.fq.gz
  └── ...

Example:

--split Path/to/split/

--bam

PATH
Use together with the --rg flag
The multiplexed BAM-FILE, as provided by the MPI EVA Core Unit containing
adapter-trimmed and overlap-merged sequencing reads
Example:

--bam Path/to/input.bam

--rg

PATH
Use together with the --bam flag
A TSV-FILE, containing library information for the demultiplexing step.
Provide the readgroups and respective primer combinations contained in the BAM FILE
Input (index.tsv):

#Index library ID     primer_P7       primer_P5
RG1   1113    1137
RG2   1114    1138

Example:

--rg Path/to/index.tsv

Optional flags

Flag

Input type

Description

--fixed

PATH
Provide a TSV file
Map extractedReads to the specified genome for given families instead of the one determinded by quicksand.
The tag is used as 'Species' name in the reports and the filenames.
Input (fixed.tsv):

Family    Species(tag)  Genome
Hominidae Homo_sapiens  /path/to/seq.fa

Example:

--fixed Path/to/fixed.tsv

--rerun
Run the pipeline in an already processed folder
Works together with the --fixed flag
Map already extracted reads of families or orders to all the species assigned in the
--fixed references file.
These records are added to the final_report file
Example:

--fixed Path/to/fixed.tsv --rerun

--taxlvl

[o,f]
Default: f
Change taxon level (family or order level) of binned sequences after KrakenUniq.
Binned reads are still mapped against the genomes of each families reference genome.

Example: Map all reads assigned to Primates to the Homo_sapiens genome
Note: For the order-level bins, Binned reads are mapped several times to different (family) genomes.
Example:

--taxlvl o

--doublestranded
Count C to T at the 5' and G to A substitutions at the 3' end of mapped sequences,
Default: Count C to T substitutions on 5' and 3' ends.
Example:

--doublestranded

Process parameters

Flag

Input type

Description

--bamfilterflag

N
For initial bam file filtering
Filter the file based on the provided SAMTOOLS FLAG (default: 1 = filter paired reads).
see HERE to find a desired filterflag
Example:

--bamfilterflag 5

--bamfilter_length_cutoff

N
For initial bam file filtering
Filter out reads below the given length cutoff (default: 35).
Example:

--bamfilter_length_cutoff 35

--krakenuniq_min_kmers

N
For metagenomic classification
Remove families from the KrakenUniq classification results with a kmer-count of less than N (default: 129).
Example:

--krakenuniq_min_kmers 129

--krakenuniq_min_reads

N
For metagenomic classification
Remove families from the KrakenUniq classification results with less than N reads assigned (default: 3).
Example:

--krakenuniq_min_reads 3

--bamfilter_quality_cutoff

N
For after the mapping step
Filter out reads with a mapping quality below the given quality cutoff (default: 25).
Example:

--bamfilter_quality_cutoff 25

--reportfilter_percentage

N
For the creation of the filtered_report.tsv file
Filter family assignments from the final_report.tsv that have a FamPercentage value of less or equal than N percent (default: 0.5).
Example:

--reportfilter_percentage 0.5

--reportfilter_breadth

N
For the creation of the filtered_report.tsv file
Filter family assignments from the final_report.tsv that have a ProportionExpectedBreadth value of less or equal than N (default: 0.5).
Example:

--reportfilter_breadth 0.8

--compression_level

[0-9]
For the BAM output processes
Set BGZF compression level (default: 0)
Example:

--compression_level 9

Profiles

Quicksand includes several profiles that can be used with the -profile flag (Be aware: only one dash -)
delimit multiple profiles by comma

Profile

Description

singularity

Use Singularity as container software

docker

Use Docker as container software

debug

Keep intermediate files in the work directory