These scripts were written for the analyis of Arabidopsis thaliana ONT sequencing data. All scripts are intended for the use with Python v2.7.

This script was used to extract the longest reads of various sequencing datasets for a combined Col-0 genome assembly.

python select_long_reads.py
--in <FULL_PATH_TO_INPUT_FILE>
--out <FULL_PATH_TO_OUTPUT_FILE>

optional:
--cut <LENGTH_CUTOFF_IN_BP>

--in specifies a gzip compressed FASTQ input file which contains the long reads.

--out specifies a gzip compressed FASTQ output file. The selected ultra long reads are written into this file.

--cut specifies a length cutoff for the selection of ultra long reads. Only reads passing this filter will be written into the output file. Default: 3001bp.

This script was used to remove 'small' structural variants from the SVIM results.

python filter_SVIM_VCF.py
--in <INPUT_VCF>
--out <OUTPUT_VCF>
--minsize <MINIMAL_SV_SIZE>[1000]

--in specifies a VCF file produced by SVIM.

--out specifies a VCF file which will include the variants passing the specified length filter.

--minsize specifies the minimal size of structural variants to be included in the output file. Default: 1000 bp.

This script is intended to generate a genome-wide distribution plot of SVIM results.

python genome_wide_distribution.py
--in <SVIM_VCF_FILE>
--ref <REFERENCE_SEQUENCE>
--fig <FIGURE_NAME_WITH_PDF_EXTENSION>

optional:
--score <INT, minimal SVIM score to consider variant>[10]

--in specifies a VCF file produced by SVIM. Analysed variants are 'DUP:TANDEM', 'INV', 'INS:NOVEL', 'DEL', and 'DUP:INT'.

--ref specifies a FASTA file which matches the supplied VCF file.

--fig specifies the figure output file. The file format is determined by the file name extension e.g. PDF, PNG, JPEG, SVG. Support for the file formats depends on the local system.

--score specifies a minimal score cutoff to filter out low quality variants.

This script is based on previous studies (10.3390/genes10090671, 10.1534/g3.119.400847). The coverage of a read mapping is plotted along the pseudochromosomes. This script is customized for the Col-0 reference genome sequence TAIR9/TAIR10.

python cov_plot2.py
--in <FULL_PATH_TO_COVERAGE_FILE>
--out <FULL_PATH_TO_OUTPUT_FILE>
--ref <FULL_PATH_TO_REFERENCE_COVERAGE_FILE>
					
optional:
--name <NAME>

--in specifies a coverage file. This TAB-separated file contains the pseudochromosome name, position, and coverage of the respective position. This file can be generated based on a BAM file using https://github.com/bpucker/MGSE/blob/master/construct_cov_file.py.

--ref specifies a FASTA file which was used to generate the read mapping. It is important that pseudochromosome names match the names in the coverage file.

--out specifies an output folder, where the figure file and the data file will be placed. This folder will be created if it does not exist already.

--name specifies a name to be included in all file names.

This script is intended to analyse the local quality along ONT reads.

python analyze_read_quality.py
--in <FASTQ_INPUT_FILE>
--info <INFO_FILE>
--out <OUTPUT_FOLDER>

--in specifies a FASTQ input file which contains long reads.

--info specifies a text file with details about the reads of interest. This file is TAB-separated with the following columns: SampleID, ReadID (need to match a read in the supplied FASTQ), Start, End, and Comment (optiona). Start and end can be used to analyse just a fraction of a read.

--out specifies the output folder.

This script is intended to remove everything except the actual contig name from the header line in a multiple FASTA file.

python reduce_contig_names.py
--in <INPUT_FILE>
--out <OUTPUT_FILE>

--in specifies a FASTA input file which contains sequences with complex header lines.

--out specifies the output folder. Sequences will be written to the output file, but header lines will be reduced to the actual contig name.

This script is intended to remove contamination from a long read assembly file. The general concept is based on previous work ( 10.1371/journal.pone.0164321, 10.3389/fmolb.2018.00062, 10.3390/genes11030274).

python clean_assembly.py
--out <OUTPUT_FOLDER>
--assembly <ASSEMBLY_FILE>
--cov <COVERAGE_FILE>
--organel <ORGANEL_SEQ_FILE>
--white <WHITE_LIST_SEQ_FILE>
--black <BLACK_LIST_SEQ_FILIE>
					
optional:
--minlen <INT, MINIMAL_CONTIG_LENGTH>
--mincov <INT, MINIMAL_READ_MAPPING_COVERAGE_FOR_FILTERING>
--maxcov <INT, MAXIMAL_READ_MAPPING_COVERAGE_FOR_FILTERING>
--wordsize <INT, WORD_SIZE_FOR_BLAST>
--cpus <INT, NUMBER_OF_THREADS_FOR_BLAST_SEARCH>

--assembly specifies a FASTA input file which contains sequences with complex header lines.

--out specifies the output folder. All output files will be stored in this folder. The folder will be created if it does not exist already.

--cov specifies a coverage file. The sequence names in this coverage file must match the sequence names in the assembly file. The average coverage per contig can be used as filter.

--organel specifies a FASTA file containing the organellar genome sequences of the species of interest or closely related species. This is used to identiy contigs belonging to these subgenomes.

--white specifies a FASTA file with white list sequences. Hits against these sequences will be used to preserve the sequences during the filter steps.

--black specifies a FASTA file with black list sequences. Hits against these sequences will be used to discard the sequences during the filter steps unless their are also hits against the white list sequences.

--minlen specifies a minimal contig length. All contigs shorter than this value will be removed in the filtering. This value should be set based on the read length distribution. Default: 100000 bp (100kb).

--mincov specifies a minimal coverage in the read mapping that is expected for all valid contigs. This filter can be used to identify artifacts with low coverage. This filter needs to be adapted to the average coverage. Default: 10.

--maxcov specifies a maximal coverage in the read mapping that is expected for all valid contigs. This filter can be used to identify and discard collapsed sequences or organellar sequences. This value should be set depending on the average coverage. Default: 200.

--wordsize specifies the '-word_size' parameter which is passed on to BLAST. Using a large value can substantially speed up the process, but decreases the sensitivity. Default: 30.

--cpus specifies the number of threads to use for BLAST. Default: 20.

Long reads allow the evaluation of existing assemblies through the inspection of reads mappings. A high number of read alignments clustered in a region can point to potential assembly errors. AEF.py analyzes the number of such read alignment ends per region to find critical regions with enriched alignment ends.

python AEF.py
--out <OUTPUT_FOLDER>
--bed <INPUT_BED_FILE> | --bam <INPUT_BAM_FILE>
--fasta <ASSEMBLY_FASTA_FILE>

optional:
--gff <GFF_FILE>
--res <RESOLUTION>[100]
--sat <SATURATION>[100]
--name <NAME>[xxx]
--relfreq <RELATIVE_ALIGNMENT_END_CUTOFF>[10]
--factor <IQR_OVER_MEDIAN_CUTOFF>[5]
--dist <EXCLUDE_DISTANCE_TO_SEQ_ENDS>[1000]
--tolerance <DIST_TO_GENE>[0]

--out specifies the output folder. All output files will be stored in this folder. The folder will be created if it does not exist already.

--bed specifies a BED input file which contains sequences with complex header lines. --bam is an alternative input option which specifies a BAM file. --in can be used to provide a BED file.

--fasta specifies a FASTA assembly file which is used to check all sequences and and to provide the output in the correct order.

--gff specifies GFF3 file which contains positions of annotated gene models. This file is used to check if potential assembly errors could affect genes.

--res specifies the resolution of this analysis. The specified window size (base pairs) is used to find enrichments of read alignment ends. Default: 100bp.

--sat specifies a saturation for the visualization. This is important to accommodate all values in one figure. The best value depends on the average coverage. Usually, a value below the average coverage should be a good choice. Default: 100x.

--name specifies a name which is used as prefix for the output file.

--relfreq specifies a minimal cutoff for the relative frequency of alignment ends in a window. Only windows with more than this relative value will be reported as potential assembly errors. The number of read alignment ends in a window is normalized to the average number of such alignment ends over all windows. Default: 10.

--factor specifies a minimal cutoff for windows to be considered as assembly errors. This factor determines how many interquartile ranges (IQRs) the value of a window needs to be above the median of all windows to consider a region. This option is slightly redundant with --relfreq. Default: 5.

--dist specifies a distance to contig/pseudochromosome ends which is masked in the analysis. No assembly errors are reported in these regions, because read alignment ends must be enriched in these regions.

--tolerance specifies the tolerated distance between a gene and the next assembly error to flag the gene as potentially effected.

python group_to_regions.py
--out <OUTPUT_FILE>
--in <INPUT_FILE>

optional:
--win <WINDOW_SIZE>[30000]

--in specifies the input file which was produced by AEF.py.

--out specifies the output file which contains the assembly error candidate regions.

--win specifies the maximal distance between initial feature that will be joined into one candidate region. Default: 30kb.

This scripts compared two given sequences (FASTA files) against each other and visualizes the results in a dot plot. The color of dots is used as a heatmap to indicate the similarity of hits. This scripts is based on previous work (Pucker et al., 2019). However, this script was adjusted to include information about genes affected by potential inaccuracies in the reference genome sequence. Additionally, the IDs of underlying BACs are included if available.

python dot_plot_heatmap.py
--out <OUTPUT_FOLDER>
--in1 <INPUT_FASTA_FILE1>
--in2 <INPUT_FASTA_FILE2>

optional:
--gff1 <GFF_FILE_TO_INCLUDE_AGIs>
--gff2 <GFF_FILE_TO_INCLUDE_BAC_IDs>
--chr <CHROMOSOME>
--start <START_POSITION>
--end <END_POSITION>

--block <SEQ_LENGTH_PER_BLOCK>
--namex <NAME_ON_X_AXIS>
--namey <NAME_ON_Y_AXIS>
--title <TITLE>
--name <PREFIX_NAME_OF_RESULT_FILE>

--show activates the display of an interactive figure
--cite will not run the script, but display the reference

--in1 specifies the first input sequence. This FASTA file must contain exactly one sequence. This sequence will be represented on the x-axis.

--in2 specifies the second input sequence. This FASTA file must contain exactly one sequence. This sequence will be represented on the y-axis.

--out specifies the output folder. This folder will be created if it does not exist already.

--gff1 specifies a GFF3 file with the Araport11 annotation to include the AGIs of genes in the region.

--gff2 specifies a GFF3 file with details about BACs in the region. This information is used to display the IDs of BACs underlying the sequence in the dot plot.

--chr specifies the name of the chromosome which harbours the investigated region.

--start specifies the start of the investigated region on the chromosome given above.

--end specifies the end of the investigated region on the chromosome given above.

--block specifies the size of blocks which are represented by a dot in the figure. [1000]

--namex specifies the name to display on the x-axis.

--namey specifies the name to display on the y-axis.

--title specifies the title to display on top of the figure.

--name specifies a prefix for the name of all output files.

--show activate the display of an interactive figure. This should not be required in most cases. [off]

--cite shows the reference of this script.

Pucker, B.+, Kleinbölting, N.+ and Weisshaar, B. (2021). Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis. doi: 10.1101/2021.03.03.433755. + both authors contributed equally

Pucker, B (2019). Mapping-based genome size estimation. bioRxiv. doi:10.1101/607390.

Pucker B, Rückert C, Stracke R, Viehöver P, Kalinowski J, Weisshaar B. Twenty-Five Years of Propagation in Suspension Cell Culture Results in Substantial Alterations of the Arabidopsis Thaliana Genome. Genes. 2019. doi:10.3390/genes10090671.

Busche M*, Pucker B*, Viehöver P, Weisshaar B, Stracke R. (2020). Genome Sequencing of Musa acuminata Dwarf Cavendish Reveals a Duplication of a Large Segment of Chromosome 2. G3: Genes|Genomes|Genetics. doi:10.1534/g3.119.400847.

Pucker, B, Holtgräwe, D, Rosleff Sörensen, T, Stracke, R, Viehöver, P, and Weisshaar, B (2016). A de novo Genome Sequence Assembly of the Arabidopsis thaliana Accession Niederzenz-1 Displays Presence/Absence Variation and Strong Synteny. PloS-ONE 11:e0164321. doi:10.1371/journal.pone.0164321.

Siadjeu C*, Pucker B*, Viehoever P, Albach D and Weisshaar B (2020). High contiguity de novo genome sequence assembly of Trifoliate yam (Dioscorea dumetorum) using long read sequencing. Genes. doi:10.3390/genes11030274.

Haak, M, Vinke, S, Keller, W, Droste, J, Rückert, C, Kalinowski, J, & Pucker, B. (2018). High Quality de novo Transcriptome Assembly of Croton tiglium. Frontiers in Molecular Biosciences, 5. doi:10.3389/fmolb.2018.00062.