Laura’s Notebook: Oly OA RNA isolation – raw data

Erica at the NWGC worked very quickly to get my samples sequenced on the NovaSeq. On Monday she pooled them and ran each pool on the Bioanalyzer. Here is the bioanalyzer report, and a note from Erica about the pool concentrations:

Here is the QC: I did dilute an aliquot of the pool down further to run the samples better. The original qubit pools were as follows and these were what was loaded on the bioanalyzer.

  • Batch1_69plex 10.966ng/ul
  • Batch2_77plex 11.649ng/ul

The diluted qubit values are:

  • Batch1_69plex 2.777ng/ul
  • Batch2_77plex 2.43ng/ul

Once the samples were finished on the NovaSeq, she sent me a summary of the run, and files for each pool with the # reads per sample:

Date Lane Lane Concentration (pM) SAV Clusters/mm^2 % Clusters PF Overall Q30 Read 1 Q30
200420 1 Batch1_69plex 270 506.12 79.29 89.73 88.93
2 Batch2_77plex

I received a link to the raw data, which is not yet demultiplexed (I will need to do that). The data is stored on Globus.org. I will need to transfer it from there to the Nightengales directory on Owl in the O. lurida folder, which is where all raw NGS data is housed for the Roberts Lab. This could be done in a few ways, but I will follow Shelly’s lead, with a few changes based on my work-at-home situation. She directed me to this notebook post, and this github issue as references. Globus endpoints are set up via a GUI, so since I am working at home I opted to tranfer the files to my external hard drive, and then to other locations.

Set up Globus personal endpoint, download data

Following these Globus setup instructions I set my laptop up as a personal endpoint on Globus. I edited my Globus settings to be able to write to my external hard drive. I then transferred the two FASTQ files with their md5 files to my external hard drive.

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Mount Owl, transfer files to Nightengales

I mounted Owl on my computer using Finder –> Go –> Connect to Server. I entered owl’s address (afp://owl.fish.washington.edu), then my username and pw.

To be continued…

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