SMRT-Portal Exercises. J Fass UCD Genome Center Bioinformatics Core Thursday April 16, 2015

Transcription

1 SMRT-Portal Exercises J Fass UCD Genome Center Bioinformatics Core Thursday April 16, 2015

2 Running SMRT-Portal in AWS see PacBio documentation We ll be running a virtual machine (VM) in the Amazon Web services Cloud (a server farm somewhere in the region you ve selected). On this VM is a web server, serving you pages created by the SMRT-Portal application.

3 Running SMRT-Portal in AWS Launch an m3.2xlarge instance using ami953fddd1. Generate or re-use a key pair - you will need it! Once running, find the public IP address (#.#.#. #), and open a browser tab with the URL: #.#.#.#:8080/smrtanalysis or #.#.#.#:8080/smrtportal

4 Running SMRT-Portal in AWS On a vanilla PacBio SMRT-Portal instance (U. S. East / N. Virginia), you would need to create one administrator account. This AMI already has one, but feel free to change the password, add non-admin accounts, etc. user: administrator pwd: 5MRT-P0rtal Note: pwd = >0 symbols, >0 numbers, >8 characters

5 Running SMRT-Portal in AWS Log in as administrator (special user), then create separate accounts if desired.

6 How I imported 8 SMRT Cells (E coli)

7 SSH to AWS instance ssh -i ~/.ssh/yourkey.pem ssh command option block (supplies private key in this case) destination (or use PuTTY)

8 PacBio Public Datasets look for Data supporting publications

9 PacBio Public Datasets look for Data supporting publications look for the first MG1655 xml & bas.h5 files

10 Enter dropbox directory cd /opt/smrtanalysis/userdata/inputs_dropbox cd command destination directory

11 Pull in data mkdir MG1655 cd MG1655 wget [xml file link] mkdir Analysis_Results cd Analysis_Results wget [bas.h5 file link, + bax.h5 s if present] command directory / destination / source

12 Import SMRT Cell data Back in SMRT Portal, click through Home (upper left), then Import and Manage (third image), then Input SMRT Cells.

13 Import another SMRT Cell (exercise)

14 SSH to AWS instance ssh -i ~/.ssh/yourkey.pem ssh command option block (supplies private key in this case) destination (or use PuTTY)

15 PacBio Public Datasets look for E. coli size selected 20kb library

16 PacBio Public Datasets Find the SMRT Cell data files tarball, and copy the link (don t download; you ll break our wireless!).

17 Feeding Data to the SMRT-Portal Back in a shell (terminal) on your instance, navigate to SMRT-Portal s input dropbox. cd /opt/smrtanalysis/userdata/inputs_dropbox wget [link] mkdir Ecoli20kb cd Ecoli20kb mkdir Analysis_Results tar -xzvf ecolik12.tar.gz

18 Feeding Data to the SMRT-Portal Back in SMRT Portal, click through Home (upper left), then Import and Manage (third image), then Input SMRT Cells.

19 Feeding Data to the SMRT-Portal Via Home, Import and Manage, and [Import] SMRT cells, get to import page. Select directory, and Scan.

20 HGAP Assembly

21 Running HGAP Click Design Job, then Create New, (deal with the design wizard - I usually select display all protocols ). You should see 9 SMRT Cells available (we just imported the 9th).

22 Running HGAP Select the RS_HGAP_Assembly.3 Protocol from the dropdown menu, enter name and (if desired) comments, select 20kb cell and click right arrowhead to add cell to the job you re designing, then Save and Start!

23 Running HGAP early results just assess reads, subreads...

24 Running HGAP Final results include pre-assembly, realigned reads, etc.

25 HGAP output Find the Polished Assembly Fasta link, right-click and Save link as (to avoid troublesome name).

26 HGAP output Notice the BAM and BAI links; these allow you to view the original reads aligned back to the assembly (e.g. in IGV).

27 Check assembly via homology Using Mauve, we ll align our assembled genome to the trusted E. coli K-12 MG1655 reference assembly, from GenBank (link).

28 Check assembly via homology Launch Mauve, then select File Align with progressivemauve. Then Add Sequence (click to add GenBank reference, then our assembly), click Align (and add a place to save output).

29 Check assembly via homology (see Mauve site for details on viewer, etc. we ll explore during Workshop)

30 Check for circularity (if appropriate) Launch Gepard, Select file specifying the polished genome assembly twice (once for horizontal, once for vertical), then create dotplot.

31 Check for circularity (if appropriate) Looks fine, right? But the overlaps will be on the size scale of the reads not visible at this scale.

32 Check for circularity (if appropriate) Use the Advanced mode, Plot tab, to specify the first ~20kb on the horizontal, and the last ~20kb on the vertical. Then Update dotplot.

33 Alignment / Resequencing Protocols

34 Align to your own reference In SMRT-Portal, go Home, then Import and Manage, then reference sequences. Select New to upload our down loaded reference (note there s also a Scan option - upload first to /opt/smrtanalysis/userdata/references_dropbox/).

35 Align to your own reference Design a job using the same reads, and the RS_Resequencing.1 protocol. Specify your uploaded reference sequence, save, and start the job. (I m using E albertii in this case, RefSeq id NZ_CP )

36 Viewing Read Alignments with IGV