Flags

Required flags

Reference Database

These flags are related to the quicksand-build output.

Flag

Type

Description

--db

PATH

A directory containing the preindexed Kraken or KrakenUniq database:

Input:

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


Example:

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

--genomes

PATH
A directory containing the indexed FASTA-FILES of the reference genomes used to build
the KrakenUniq database. Format genomes/${family}/${species}.fasta
Input:

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


Example:

--genomes Path/to/refseq/genomes

--bedfiles

PATH
A directory containing dustmasked BED-FILES for all the reference genomes
Format masked/${species}_masked.bed
Input:

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


Example:

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

The input for quicksand is a directory with user-supplied files in BAM or FASTQ format. Adapter-trimming, overlap-merging and sequence demultiplexing need to be performed by the user prior to running quicksand. However, quicksand also implements a BAM demultiplexing software. This software is working for the bam-files provided by the MPI EVA Core Unit.

In these cases, quicksand can be used with the --bam PATH and --rg PATH flags as alternative to the --split PATH parameter.

Flag

Type

Description

--split

PATH
Default input method!
A directory containing demultiplexed, adapter trimmed (and overlap-merged) BAM or FASTQ files
Input:

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

Example:

--split Path/to/split/

--bam

PATH
Use in combination with the --rg flag
A 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 in combination 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

Type

Description

--fixed

PATH
Provide a TSV file
Map extractedReads (binned sequences) of detected families to the reference genomes listed, instead of the ones determinded by quicksand.
The 'Tag' is used as 'Species' name in the final summary reports and filenames.
Input (fixed.tsv):

Family    Species/Tag  Genome
Hominidae Homo_sapiens  /path/to/seq.fa
Hominidae Other_cool_hominin  /path/to/other_cool_hominin.fa

Example:

--fixed Path/to/fixed.tsv

--fixed_bedfiltering
Use in combination with the --fixed flag
Set this flag to run dustmasking and bedfiltering for the --fixed references as well.

Example:

--fixed Path/to/fixed.tsv --fixed_bedfiltering

--rerun
Rerun quicksand in an already processed folder
Works together with the --fixed flag
Map already binned reads of families/orders to the reference genomes listed in the
--fixed TSV file.
The analysis records are added to the existing final_report file
Example:

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

--taxlvl

[o,f]
Default: f
Change the taxonomic level for binning sequences after KrakenUniq classification (family or order level).

Example:

--taxlvl o

--doublestranded
Count C-to-T substitutions at the 5' and G-to-A substitutions at the 3' end sequence alignments,
Default: Count C-to-T substitutions on both the 5' and 3' ends.
Example:

--doublestranded

Process parameters

Flag

Type

Description

--bamfilterflag

N
For initial BAM-file filtering
Filter each BAM-file based on the provided SAMTOOLS FLAG (default: 1 = filter paired reads).
see HERE to find desired filterflags
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 removal of KrakenUniq background-identifications
Remove families from the KrakenUniq classification results with an unique kmer-count of less than N (default: 129).
Example:

--krakenuniq_min_kmers 129

--krakenuniq_min_reads

N
For removal of KrakenUniq background-identifications
Remove families from the KrakenUniq classification results with less than N reads assigned (default: 3).
Example:

--krakenuniq_min_reads 3

--bamfilter_quality_cutoff

N
Filter BAM files adter the BWA mapping step
Remove mapped sequences 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

dont delete intermediate files in the work directory after successful quicksand execution