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.
- 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
- Reference sequence DH10B_NC010473.gbk
- Launch SeqMan NGen and choose New Assembly.
- Choose Variant Analysis / Resequencing on the left, then Whole genome under NGS-Based on the right.
- In the Reference Sequence screen, press Add and add the file DH10B_NC010473.gbk. Click Next.
- 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.
- In the Assembly Options screen, click Next.
- 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.
- In the Assembly Output screen:
- Type “CNV” into the Project Name text box. Use the Browse button to specify a Project Folder for your assembly output files. Click Next.
- 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.
- 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.
- 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.
- After being informed that assembly has finished, click Next.
- From the Project Report screen, click Analyze and compare variants to open the project in ArrayStar.
- After ArrayStar opens, Use File > Save Project to save the project as CNV.astar.
- 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.
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