We began working with the VolTRAX2 today. I’m excited to use it for library preps along with the Miroculus Miro. Tonight I watched the NCM 2022 Masterclass session by Matt Attreed on “How to generate and call variants.” Attreed is a technical applications scientist (bioinformatics) with Oxford Nanopore Technologies. They began by providing an introduction to the resources available on the ONT Community pages and resources. Applications, tools, and best practices are listed. Attreed mentioned there is a page dedicated to the raw reed accuracy of Oxford Nanopore Technologies (ONT). Attreed explained that raw reads are QC assessed, filtered, and converted to a BAM file that is filtered to generate an answer. Attreed presented workflows available by ONT. All analyses start with MinKNOW. Attreed noted that MinKNOW can do basecalling, demultiplexing, and assembly. EPI2ME and EPI2ME Labs are “point-and-click” tools. EPI2ME allows for real-time analysis by uploading data as it is generated. EPI2ME Labs run on local hardware, and the minimum hardware requirement will depend on the workflows. EPI2ME Labs has 13 open-source workflows and requires Docker. Workflow parameters can be modified through a graphical interface. Attreed then spoke about reference formats in FASTA format. The reference sequence you use can be obtained from public databases. Reads are assembled into contigs which are then combined into scaffolds. Pore-C sequencing data and ultra-long reads can be used to create better assemblies. De novo assembly may need a polishing to resolve misassemblies caused by poor read alignment during assembly. For example, Attreed had a flow chart going from raw reads, to filtered reads through QC, to assembly to create a draft FASTA file, to polishing to create a consensus FASTA, to annotation for a genome to explore. Coverage is important. Attreed recommended assembling FASTQ files with Flye. The assembly can be polished using Medaka to output a FASTA file. This workflow is available in EPI2ME Labs. Attreed noted that reads can be assembled from a metagenomic sample too. This process can allow for the assembly of genomes (if possible) for identification of organisms. Attreed and ONT recommend using MetaFlye for assembly followed by polishing with Medaka, and this workflow is available in EPI2ME Labs. I want to install EPI2ME Labs on a workstation. Reads from Nanopore sequencing can also be used for the assembly of amplicons and transcriptomes, an application I had not considered.
Attreed explained that an alignment can be generated by taking reads and filtering them, aligning to a reference sequence to create a BAM file. Differences can be detected to generate a VCF file: variant call format with the positions and information about the variations. Attreed noted that there is a human variation workflow available in EPI2ME Labs. There is also a workflow for calling base modifications. The reads are aligned with minimap2, Clair3 is used to call SNV, and phasing is performed with WhatsHap to create a VCF output file. Attreed explained that there are several types of structural variants such as deletions, duplications, duplex, translocation, inversion, and insertion. To call structural variants, ONT recommends using Sniffles2 and then filtering variants to create a VCF file. Attreed noted that variant detection is possible in other organisms and workflows for SNV, SV, and methylation detection are available.
Nanopore sequencing allows for sequencing of transcripts, including cDNA and native RNA. EPI2ME Labs has a workflow for transcriptomics! This workflow can also perform de novo based transcriptome assemblies when a reference is not available. With PromethION sequencing, single-cell analysis and spatial resolution are available. The workflow seems to generate several different types of analyses. I am now eager to try EPI2ME Labs and begin learning about the workflows we can take advantage of for class datasets and undergraduate research projects.
