Genome Portal Application Data

DNA Extraction

A high-quality DNA extraction is the critical starting point to creating a complete reference-grade genome. Yet, the quantity and quality of DNA extracted from different microbial strains can vary due to the inherent differences in cell wall composition. That’s why ATCC uses several proprietary protocols that are dependent on the species being analyzed to obtain high-molecular-weight extractions from our products.

Figure 1. Assessment of quality and quantity of extracted genomic DNA. Fragment size graph obtained from the Agilent Fragment Analyzer platform.

Sequencing

The advancement and accessibility of next-generation sequencing (NGS) technologies have rapidly transformed microbiological research; however, the complexity of these technologies pose significant challenges with regard to the introduction of biases. Therefore, to generate the best quality sequencing data for our genome assemblies and to reduce NGS biases, we sequenced a single extraction of high-molecular-weight DNA on both Illumina and Oxford Nanopore sequencing platforms. The resulting data were then analyzed through our standardized workflow.

Figure 2. ATCC’s bacterial genome sequencing quality control (A) substantially improves the quality of Illumina reads, and (B) improves the length distribution of reads from the Oxford Nanopore Technologies platform. This approach ensures that the longest, highest-quality reads are used for assembly. The dashed line indicates the quality score cutoff used for each sequencing technology.

Genome Assembly

Accuracy is essential when it comes to assembling a reference-grade genome, which is why we used a hybrid assembly approach that generates accurate basecalls via short reads while scaffolding over low-complexity regions with long reads. To ensure that researchers have access to the highest-quality material possible, we only publish genomes assembled as a single contig to the genome portal.

Figure 3. Pairwise comparisons between select assembled ATCC genomes and their publicly available counterpart for a variety of assembly metrics. ATCC genomes show comparable or better assembly metrics than publicly available genomes. CDSs is coding sequences; N50 is the size of the shortest contig when 50% of the genome is contained in contigs of the same size or larger.

Figure 4. MUMmer alignment of ATCC de novo genome assembly of ATCC^® 12228™ versus the publicly available genome assembly GCA_002215535.1 and plasmid alignments. Results are indicative of substantial structural variation and no complete matching plasmids between assemblies.