--- title: "Short Read Eukaryotic Analysis" canonical: "https://help.biobam.com/space/OED0324/3525083671/Short%20Read%20Eukaryotic%20Analysis" format: markdown --- # Introduction Genome analysis of *Botryotinia fuckeliana* (anamorph *Botrytis cinerea*) strain BcDW1. ### Dataset Description Botrytis cinerea (teleomorph Botryotinia fuckeliana) is an opportunistic pathogen with a broad host range and is particularly aggressive on fleshy fruit, such as tomato, strawberries, and grape berries. DNA from an axenic BcDW1 culture was extracted using a modified cetyltrimethylammonium bromide (CTAB) method, and 6.9 Gb of Illumina sequence reads was generated to achieve >100× coverage of the genome. - Organism: *[Botryotinia fuckeliana](https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=1290391&lvl=3&lin=f&keep=1&srchmode=1&unlock)*[ (](https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=1290391&lvl=3&lin=f&keep=1&srchmode=1&unlock)*[Botrytis cinerea ](https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=1290391&lvl=3&lin=f&keep=1&srchmode=1&unlock)*[BcDW1).](https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=1290391&lvl=3&lin=f&keep=1&srchmode=1&unlock) - Instrument: * *Illumina HiSeq 2000. - Layout: Paired-end. ### Publication [Blanco-Ulate B, Allen G, Powell AL, Cantu D. Draft Genome Sequence of Botrytis cinerea BcDW1, Inoculum for Noble Rot of Grape Berries. Genome Announc. 2013 May 23;1(3):e00252-13. doi: 10.1128/genomeA.00252-13. PMID: 23704180; PMCID: PMC3662820.](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3662820/)
Abstract Botrytized wines are produced from grape berries infected by Botrytis cinerea under specific environmental conditions. Here, we report the draft genome sequence of B. cinerea BcDW1, a strain isolated from Sémillon grapes in Napa Valley in 1992 that is used with the intent to induce noble rot for botrytized wine production.
### Original Data - NCBI BioProject: [PRJNA188482](https://www.ncbi.nlm.nih.gov/bioproject/PRJNA188482). - SRA Experiment: [SRR680162](https://www.ncbi.nlm.nih.gov/sra/?term=SRR680162). - NCBI Assembly: [GCA_000349525.1](https://www.ncbi.nlm.nih.gov/assembly/GCA_000349525.1). # Bioinformatic Analysis ## 1- DNA-Seq *de novo* Assembly ### Application [DNA-Seq ](https://biobam.atlassian.net/wiki/spaces/OBD/pages/2489581675)*[de novo](https://biobam.atlassian.net/wiki/spaces/OBD/pages/2489581675)*[ Assembly](https://biobam.atlassian.net/wiki/spaces/OBD/pages/2489581675) (ABySS). ### Input - Illumina sequencing data in FASTQ format ([SRR680162_1.fastq.gz](https://drive.google.com/file/d/1cNPPiJ0ZS12GcOkCfrq7u1te9KJ1QQtS/view?usp=sharing) and [SRR680162_2.fastq.gz](https://drive.google.com/file/d/18XIyOuo2ON15DCX9hzl8a4FRRsyn0coz/view?usp=sharing)). ### Parameters - Input Reads: [Paired-End] SRR680162_1.fastq.gz and SRR680162_2.fastq.gz - Upstream Files Pattern: _1 - Downstream Files Pattern: _2 - Use Additional Data: false - K-mer Size: 64 - Use Paired de Bruijn graph: false - Minimum Alignment Length: 40 - Hash Functions: 1 - K-mer Count Threshold: 2 - Unitigs Fasta: unitigs.fasta - Contigs Fasta: contigs.fasta - Scaffolds Fasta: scaffolds.fasta - Save Graph Files: true ### Execution Time 70-90 minutes. ### Output - [unitigs.fasta: FASTA file containing assembled unitig sequences.](https://drive.google.com/file/d/1CpPX58UaxHtpevqkiLmemjoDP5HQSxra/view?usp=sharing) - [contigs.fasta: FASTA file containing assembled contig sequences.](https://drive.google.com/file/d/1StK_RFeZNjLPMJET8u6tEXca2qbPYdjA/view?usp=sharing) - [scaffolds.fasta: FASTA file containing assembled scaffold sequences.](https://drive.google.com/file/d/11uxQDniGB4X4XFPowxxkDo8b3h_680cK/view?usp=sharing) - [report_abyss.box: Report about DNA-Seq de novo Assembly results.](https://drive.google.com/file/d/1DWSGCO1let9-29IW8gFlMkpSOYaeN87q/view?usp=sharing) - [nx_plot.box: Line chart about DNA-Seq de novo Assembly results.](https://drive.google.com/file/d/1MQ1dGS5BV_SWbE64qc2Vuu9zF8jtfhqr/view?usp=sharing) - [assembly-contigs.dot: Text file containing the contig overlap graphs in the GraphViz DOT syntax.](https://drive.google.com/file/d/1QAncdBYWlmOpiGns8ONKESfxxE4zCW4S/view?usp=sharing) - [assembly-scaffolds.dot: Text file containing the scaffold overlap graphs in the GraphViz DOT syntax.](https://drive.google.com/file/d/1cJh3Bq8r_L6yO4iKAkpSzE3qCq32KzNo/view?usp=sharing) ## 2- Repeat Masking ### Application [Repeat Masking](https://biobam.atlassian.net/wiki/spaces/OBD/pages/618594328). ### Input - [Assembled genomic scaffolds](https://drive.google.com/file/d/11uxQDniGB4X4XFPowxxkDo8b3h_680cK/view?usp=sharing) in FASTA format (from the [1- DNA-Seq de novo Assembly](https://biobam.atlassian.net/wiki/spaces/OED0324/pages/3525083671/Short+Read+Eukaryotic+Analysis#1--DNA-Seq-de-novo-Assembly) step). ### Parameters - Input Sequences: scaffolds.fasta - Search Engine: RMBlast - Repeat Database: Dfam Consensus - Species: 40559 Botrytis cinerea - Speed/Sensitivity: Default - Masking Options: Soft masking (lowercase) - Apply Divergence Cutoff: false - Only Alu elements: false - Type of repeat: interspersed,simple_low - Not mask RNA genes: false - Output FASTA: masked_sequences.fasta ### Execution Time 10-15 minutes. ### Output - [masked_sequences.fasta: FASTA file containing repeat masked sequences.](https://drive.google.com/file/d/1826W-XRjHoMH0Bjhmf_PUGa3C5VqzXLx/view?usp=sharing) - [scaffolds_gff.box: GFF project containing repeat coordinates.](https://drive.google.com/file/d/1_hr4gHzlA_5ayeq5fNMA__IfYOhcR9bH/view?usp=sharing) - [report_repeat_masking.box: Report about Repeat Masking results.](https://drive.google.com/file/d/1UDmpe2y_580gAOOysb4yUqmFbVZN6zCS/view?usp=sharing) - [repeat_type_distribution.box: Pie chart showing the abundance of each type of detected repeats.](https://drive.google.com/file/d/1IEAAh41FC8Pq9ib5sCVPk2VH58g2ctec/view?usp=sharing) ## 3- Gene Finding ### Application [Eukaryotic Gene Finding by AUGUSTUS](https://biobam.atlassian.net/wiki/spaces/OBD/pages/2127462414). ### Input - [Repeat masked sequences](https://drive.google.com/file/d/1826W-XRjHoMH0Bjhmf_PUGa3C5VqzXLx/view?usp=sharing) in FASTA format (from the [2- Repeat Masking](https://biobam.atlassian.net/wiki/spaces/OED0324/pages/3525083671/Short+Read+Eukaryotic+Analysis#2--Repeat-Masking) step). ### Parameters - Input Sequences: masked_sequences.fasta - Closest Species: Botrytis cinerea [Fungi - Ascomycota - Leotiomycetes] - Strand: Both Strands - Allowed Gene Structure: Partial - Output Genomic Features: introns,start,stop - Ignore Strand Conflicts: false - UTR Prediction: false - No In-frame Stop Codons: false - Stop Codons Excluded From CDS: false - Softmasked Sequences: true - Sample: 100 - Alternatives From Sampling: false - Gene Finding Mode: Ab Initio Prediction ### Execution Time 5-10 minutes ### Output - [cds.box: Sequence project containing predicted gene sequences.](https://drive.google.com/file/d/1i4_z88UnmRzSRn4qKwVBke5VDOwURRvR/view?usp=sharing) - [protein.box: Sequence project containing predicted protein sequences.](https://drive.google.com/file/d/15VLHIAUQ5hOB2XsJJvqV3DeKxt0gsenH/view?usp=sharing) - [coordinates.box: GFF project containing predicted gene coordinates.](https://drive.google.com/file/d/1Qh3H_u8m0pFxS-zQVyHDBCwvxG-7Q0vy/view?usp=sharing) - [report_eukaryotic_gene_finding.box: Report about Eukaryotic Gene Finding results.](https://drive.google.com/file/d/1urtUeFTeQF9gvpWOc_JamQdUj-OBMInp/view?usp=sharing) - [cds_length_distribution.box: Bar chart showing the length distribution of the predicted genes.](https://drive.google.com/file/d/19uhY8F5LZTh7-w9g6ptLJqCD0bqd6MhR/view?usp=sharing) ## 4- BLAST & InterProScan ### Application [CloudBLAST](https://biobam.atlassian.net/wiki/spaces/OBD/pages/3335684097) & [InterProScan Annotation](https://biobam.atlassian.net/wiki/spaces/OBD/pages/598048967). ### Input - Predicted gene sequences in an OmicsBox project (from the 3- Gene Finding step). ### Parameters ### Execution Time ~ 1 hour. ### Output - [blast_ips_protein.box: Project containing predicted protein sequences along with Blast and IPS results.](https://drive.google.com/file/d/1Q4yIjR4wpq0c43l3N6zn9BKPrWmotyUg/view?usp=sharing) ## Workflow ![image](media://f5a4f77e-dd7d-439b-8c98-3416994d1147)