Part A: Setting up the CNV project in SeqMan NGen

In this part of the tutorial, you will use the SeqMan NGen wizard to import data and run the assembly. You will then press a button to open the results in ArrayStar.

1. Download T3_CNV.zip (1.4 GB) and extract the contents to any convenient location (e.g., your computer’s desktop). The folder contains the following sequences:
   
   
   • Reference sequence DH10B_NC010473.gbk
     
     
   • Paired end sample sequences SRR1284938_1.fastq and SRR1284938_2.fastq

2. Launch SeqMan NGen and choose New Assembly.

3. Choose Variant Analysis / Resequencing on the left, then Whole genome under NGS-Based on the right.

4. In the Reference Sequence screen, press Add and add the file DH10B_NC010473.gbk. Click Next.

5. In the Input Sequences screen, press Add and add the paired reads SRR1284938_1.fastq and SRR1284938_2.fastq. Use the Experimental setup menu to choose Single sample, then click Next.

6. In the Assembly Options screen, click Next.

7. In the Analysis Options screen, choose Haploid, since this is a bacterial genome. Check the box next to Detect CNVs and keep the default method of RPK_CN. Click Next.

8. In the Assembly Output screen:
   
   
   1. Type “CNV” into the Project Name text box. Use the Browse button to specify a Project Folder for your assembly output files. Click Next.

9. In the Run Assembly Project screen, note that the Estimated coverage is 375X. A coverage of 50-100X is adequate and additional coverage simply slows down the assembly.

10. To reduce coverage, click Assembly Options on the left. Check the box next to Maximum total reads and type in 1600000 (16 followed by 5 zeros). Then click Run Assembly Project to return to that screen. Note that the Estimated coverage is now near 50X.

11. Note the recommendation to assemble on your local computer or on the cloud and click the corresponding link to begin assembly. Local assembly should take approximately 30 minutes.

12. After being informed that assembly has finished, click Next.

13. From the Project Report screen, click Analyze and compare variants to open the project in ArrayStar.

14. After ArrayStar opens, Use File > Save Project to save the project as CNV.astar.

15. Close the SeqMan NGen project by clicking the Finish button and confirming you would like to close the application.

Proceed to Part B: Finding a putative duplication in the reference sequence using ArrayStar.