vectorQC: 'A pipeline for assembling and annotation of vectors'
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DNA vectors are widely used in molecular cloning, gene engineering, studies of gene expression and other applications. Sequence validation of a vector DNA is a crucial quality control step before using the vector. With the cost of sequencing rapidly decreasing it becomes cost-effective to ensure the vectors quality using high-throughput sequencing and bioinformatics analysis. VectorQC is an automatic pipeline for quality control of a collection of sequenced DNA vectors. The pipeline is built using the NextFlow framework and is distributed with the Docker container, which makes the pipeline easy to install, modify, and re-use on any Unix-compatible OS on a computer, cluster or cloud
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vectorQC: 'A pipeline for assembling and annotation of vectors'
- 1. Luca Cozzuto Bioinformatics Core Facility vectorQC A pipeline for assembling and annotation of vectors
- 2. Background A vector is a DNA molecule used as a vehicle to carry foreign genetic material into a cell, where it can be replicated and/or expressed. The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector.
- 3. Background Vector Host cell A vector is a DNA molecule used as a vehicle to carry foreign genetic material into a cell, where it can be replicated and/or expressed. The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector.
- 4. Background Vector Host cell Amplification (cloning vector) A vector is a DNA molecule used as a vehicle to carry foreign genetic material into a cell, where it can be replicated and/or expressed. The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector.
- 5. Background Vector Host cell Amplification (cloning vector) Expression (expression vector) A vector is a DNA molecule used as a vehicle to carry foreign genetic material into a cell, where it can be replicated and/or expressed. The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector.
- 6. Background A vector is composed of different elements: • Origin of replication • Cloning sites: one or more targets for restriction enzymes The pBR322 plasmid • Reporter genes: genes that activate / inactivate their function after successful insertion and colour the positive colonies • Antibiotic resistance: for selecting only the colonies containing the vector • Promoter • … Source: wikipedia
- 7. The problem Nowadays vectors are considered a basic tools in biotechnology and having a library of vector in a lab / facility is quite common. After each year there is an increase of the risk of mis-labelling, construct degradation, contamination. Having a quality control of the integrity of the vectors backbone and of the inserted DNA could help in avoiding wasting of time and money and in reducing errors.
- 8. Solution Biomolecular Screening & Protein Technologies Unit Genomics Unit Bioinformatics Unit
- 10. Solution Massive sequencing Pool of vectors Analysis Reproducible pipeline
- 11. Solution Massive sequencing Pool of vectors Analysis Reproducible pipeline Result Report and map of each vector Database
- 12. The pipeline: vectorQC Fragmented DNA Scaffolds / whole constructs Quality trimming and assembly
- 13. vectorQC Fragmented DNA Scaffolds / whole constructs Quality trimming and assembly Annotation of features DB of features + list of inserts Annotations
- 14. Fragmented DNA Scaffolds / whole constructs Quality trimming and assembly Annotation of features DB of features + list of inserts Annotations Generating maps Generating report and sequences vectorQC
- 15. Quality control and trimming • FASTQC: QC of initial and trimmed reads • Skewer: trimming the raw reads. vectorQC
- 16. Quality control and trimming • FASTQC: QC of initial and trimmed reads • Skewer: trimming the raw reads. Read assembly • Flash: merging of overlapping reads (optional) • SPAdes: assembly that is corrected with a custom script for addressing the circularity • Custom script: to randomly join the scaffolds in a single molecule vectorQC
- 17. Quality control and trimming • FASTQC: QC of initial and trimmed reads • Skewer: trimming the raw reads. Read assembly • Flash: merging of overlapping reads (optional) • SPAdes: assembly that is corrected with a custom script for addressing the circularity • Custom script: to randomly join the scaffolds in a single molecule Annotation • Blast: annotating features and eventually detecting the DNA insert. • Restrict (Emboss): for detecting restriction enzyme sites • Circular Genome Viewer: for generating the maps • MultiQC: for collecting the results in a comprehensive report vectorQC
- 18. Available resources • Database of features: from Plasmapper tool, but can be expanded • Database of restriction enzyme: REBASE Custom resources • Insert list: custom fasta file with the name of the inserts vectorQC
- 19. Available resources • Database of features: from Plasmapper tool, but can be expanded • Database of restriction enzyme: REBASE Custom resources • Insert list: custom fasta file with the name of the inserts https://github.com/biocorecrg/vectorQC vectorQC
- 20. Available resources • Database of features: from Plasmapper tool, but can be expanded • Database of restriction enzyme: REBASE Custom resources • Insert list: custom fasta file with the name of the inserts https://github.com/biocorecrg/vectorQC vectorQC
- 21. vectorQC
- 22. vectorQC
- 23. vectorQC
- 24. Good practices
- 26. Good practices Docker image in dockerhub with automatic buildings
- 27. Next developments • Improving the assembly: removing the low covered contigs • Comparison with reference: if provided we should check the concordance of the contigs with the reference • Detection of variants: SNP / Indel calling against the reference if provided https://github.com/biocorecrg/vectorQC
- 28. Thank you! Toni Hermoso Pulido Julia Ponomarenko Sarah Bonnin Jochen Hecht (Genomics Unit) Carlo Carolis (BS&PT Unit)