The Sanger Validation workflow allows you to co-assemble non-Sanger and Sanger data in SeqMan NGen, and then view the results in SeqMan Pro. This workflow will save data in .sqd format as long as there are fewer than 10M reads.

There are several circumstances where you may wish to create this type of hybrid assembly:

  • To close gaps in genome workflows.
  • To use Sanger reads to validate variants in non-Sanger (usually Illumina) assemblies.

Example: In order to look for a likely disease-causing variant, a gene panel assembly is run using next-gen data (e.g., Illumina, Ion Torrent). Once candidates are found, the areas around the variants are resequenced with Sanger. The Sanger trace data can then be used to confirm the variants discovered in the NGS data.

  • To test for errors in synthesized products.

Example: A high-volume facility for making synthesized DNA fragments or clones normally uses next-gen data for sequencing. But before sending synthesized products (e.g., plasmid inserts) to clients, they use Sanger to investigate and resolve errors. The Sanger Validation workflow is used to differentiate between synthesis errors and NGS sequencing errors.

In this tutorial, you will use an E. coli data set consisting of a set of 498 Sanger reads, and a set of paired-end Illumina reads with a depth of 32.

Begin with Part A: Setting up and running the assembly in SeqMan NGen.

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