This first part of Tutorial 5 begins with a Validation Control Accuracy assembly in SeqMan NGen.

  1. Click to download the data folder data folder Then extract the contents to any convenient location (e.g., your computer’s desktop). The folder contains the following sequences:

    • The genome template package Homo sapiens GRCh37.p13.

    • The VCF file NIST_RTG_PlatGen_merged_highconfidence_v0.2_Allannotate.vcf

    • The BED file Sample.bed

    • The paired end sample sequences NA12878_R1_001.fastq and NA12878_R2_001.fastq
  1. Launch SeqMan NGen.
  1. In the Begin Project screen, click the image for Assemble on local computer or Assemble on the DNASTAR cloud.
  1. In the Choose Assembly Workflow screen, select Exome and gene panel and press Next.
  1. From the Choose Assembly Type screen, pick Variant calling accuracy test and choose a location for temporary files using the Browse button. Click Next.
  1. In the Input Reference Sequences screen:

    1. Click the Download Genome Package button. Select Homo sapiens assembly GRCh37.p13 and press Select.

    2. Choose a name and location in which to save the package and press Save. This file is approximately 4GB in size and will require some time to download. When you see the message “Download Complete,” press OK.

    3. Click the Browse button to the right of VCF file, near the bottom of the screen. Select the file NIST_RTG_PlatGen_merged_highconfidence_v0.2_Allannotate.vcf and click Open.

    4. Click the Browse button to the right of Targeted regions file. Select the file Sample.bed and click Open.

    5. Press Next.
  1. In the Input Sequence Files and Define Experiments or Individual Replicates screen:

    1. Set the Read technology to Illumina. Note that the paired-end data box is now checked.

    2. Click the Add button. Navigate to and select both NA12878[…]fastq files. Click Open.

    3. In the Set Pair Information box, leave the default value of 500 and click OK.

    4. Press Next.
  1. In the Assembly Options screen:

    1. Next to Variant detection mode, select Diploid, as this is a human sample.

    2. Next to Gender, choose Female.

    3. Press Next.
  1. In the Assembly Output screen:

    1. Type “NA12878” into the Project Name text box. This name will be assigned to all output files, including the finished assembly.

    2. Use the Browse button to specify a Project Folder for your assembly output files. For local users, an alternative way to select a location is to drag and drop a folder from the file explorer onto the Project Folder row.

    3. Click Next.
  1. In the “Your assembly is ready to begin” screen, press Start Assembly to begin the assembly. The assembly will take ~30 minutes.

As soon as assembly begins, proceed to Part B: Running the validation analysis in ArrayStar.

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