Templated long-read workflow (ARTIC Amplicon)

Starting with the release of Lasergene 17.3 (2021), SeqMan NGen supports both templated and de novo assembly of long-read data from Oxford Nanopore Technologies (ONT), Pacific Biosciences (PacBio) CLR and PacBio HiFi. The templated long-read workflow, known as “ARTIC Amplicon” workflow within SeqMan NGen, is intended solely for viral genome analysis, and is typically used to monitor the evolution of viral strains such as SARS-CoV-2. This workflow has different starting points depending on the data available.

What is ARTIC? The ARTIC network developed a PCR primer set for the rapid amplicon based sequencing of SARS-CoV-2. When you send your viral genome samples to a core facility that runs the ARTIC (or similar) protocol, the result can be either type of data in the bullet points below.

If your samples are sequenced and assembled into an accurate draft genome by your sequencing core (whether the ARTIC protocol was used or not), the analysis in the second bullet point would be your usual starting point.

• An already-assembled draft genome for each experimental sample (most common) – In this case, you will use MegAlign Pro as described below to multiply align the experimental draft genomes to a pool of known viral genomes using the MAFFT alignment algorithm. In DNASTAR’s in-house tests, this algorithm aligned 10,000 SARS-CoV-2 genomes in under twelve minutes. You can then analyze relationships between samples using the distance table and/or by creating phylogenetic trees.

• Raw long read data for each experimental sample – In this case, you will start by using SeqMan NGen to perform reference-guided assembly and variant calls of the long read amplicon sequence data. You can then analyze individual alignments in SeqMan Ultra or compare variants across samples in ArrayStar. You can also output consensus sequences from SeqMan Ultra and use them in the same MegAlign Pro workflow mentioned above.

Starting with raw long-read data:

1. Launch SeqMan NGen. From the Workflow screen, choose the Variant Analysis / Resequencing tab and then select ARTIC Amplicon. Click Next.

2. Follow the steps in the SeqMan NGen wizard to input data and select assembly options. In the Input Sequences screen, use the Read technology menu to specify Nanopore, PacBio CLR, or PacBio HiFi. Most researchers running this workflow will want to specify the Experiment setup in this screen as Single or Multi-sample. These options will assemble each experimental sequence against the template as a separate assembly. The output will be a set of draft genomes in .assembly format.

3. In the Run Assembly Project screen, choose whether to assemble on your local computer or on the cloud.

4. Once assembly is complete, proceed to the wizard’s Assembly Summary screen.
   
   
   • To compare multiple samples using ArrayStar, press the Analyze and compare variants button.
     
     
   • To open one or more draft assemblies in SeqMan Ultra, press the Open assembly button. In SeqMan Ultra you can view the assembled draft genomes in graphical format with variants shown in a unique color scheme, or examine and filter individual variants in the Variants table. For example, if you were studying SARS-CoV-2, you could use filtering to locate all non-synonymous variants in the spike protein. You can also use SeqMan Ultra to export consensus sequences for the strains of interest and then analyze these in MegAlign Pro as described below.

Starting with draft genomes or already-assembled contigs:

1. Launch MegAlign Pro. From the Welcome screen, choose New blank alignment project.

2. Use File > Add Sequences to add the known genomes for the virus of interest and the experimental draft genomes from the core facility.

3. Use Align > Align Using MAFFT to perform a multiple alignment.

4. Use the Distance view and Tree view to see how the novel sequences compare to the templates.

For example, in the Distance table, you could select one or more template rows, then choose Distance > Order Sequences by Distance from Selection to move variants to the bottom of the table. The Distance area of the Style panel could be used to apply a heat map color scheme that further distinguishes sequences that most vary from the chosen reference.