Submitting NGS 16SrRNA sequences to NCBI SRA

Backing up raw sequences from mim_c

learning journey
code
Author

Sarah Tanja

Published

December 13, 2024

Modified

April 2, 2025

Background research

Log in to NCBI

Navigate to the NCBI Submission Portal: https://submit.ncbi.nlm.nih.gov/

Log-in through a partner sign-in, or sign-up directly with NCBI to create an account.

I signed in using my existing ORC-ID account, and authorized NCBI to access my ORCID record.

Using my orcid, I created a new NCBI account.

I was prompted to update my profile and confirm my primary email address in order to be able to submit to SRA.

Once at the Submission portal and logged-in, select your sequence type (16S rRNA, genome, ITS, SRA)…. For RNA-seq click on SRA to continue to the Sequence Read Archive (SRA) submission portal: https://submit.ncbi.nlm.nih.gov/about/sra/

Click SUBMIT to start the submission process.

Click New Submission

There are 5 steps to submission:

1. Submitter contact info (fill this out)

2. General Info

Click CONTINUE

3. Project Info

  • Project Title

  • Public description

  • Grants

4. Biosample Type

  • Typed in Montipora capitata

  • Under displayed NCBI packages I selected ‘Invertebrate’

  • Clicked CONTINUE

5. Biosample Attributes

  • Use built-in table editor

    The following are the columns:

  • Sample Name

    • Here I put the sample_id

  • Sample title

    • Same as Sample Name

  • BioProject accession : left blank

  • Organism

  • isolate : left blank

  • breed : not applicable

  • host

    • Montipora capitata

  • isolation source

    • I put ‘developing coral embryos from (n) distinct bundle-bundle crosses’

  • collection date

    • date each cross was culled

  • geographic location

    • USA:Oahu, Kaneohe Bay

  • tissue

    • whole embryo

  • age

    • hours post fertilization (4, 9, 14)

  • altitude : blank

  • biomaterial provider

    • Dr. Padilla-Gamiño, University of Washington, School of Aquatic and Fishery Sciences, Box 355020, Seattle, WA 98195

  • collected by

    • Sarah Tanja, Katherine Lasdin

  • depth : blank

  • development stage

    • cleavage, prawn chip, or early gastrula

  • broad-scale environmental contex

    • coral reef biome

  • host tissue sampled : blank

  • identified by: blank

  • latitude and longitude

    • 21.431972 N 157.787472 W

  • sex : blank

  • specimen voucher : blank

  • temperature : blank

  • sample description

    • description of pooled samples

  • PVC leachate treatment concentration level

  • PVC leachate treatment concentration mg/L

  • pooled crosses

Click CONTINUE

6. SRA Metadata

I provided metadata with the built-in table editor, and copied columns from my metadata google sheet into the NIH Submission Portal.

7. File Upload

SRA file transfer protocol directions here

Expand the FTP instructions section to follow instuctions mput

From the R terminal in the directory where the fastq.gz and fastq.gz.md5 files are located…

Start your FTP session:

`ftp ftp-private.ncbi.nlm.nih.gov`

Log in with your username and password provided in the FTP instructions drop-down

cd into your uploads directory as described in the FTP instructions drop-down

make a new directory for your upload (I use the same name as the git repo for the project)

  • Disable the interactive prompt by typing:

    prompt

  • Use mput to upload all files:

    mput *fastq*

  • Each file will show Transfer complete as it is uploaded

  • When finished, exit the FTP session:

    bye

Wait 10 minutes

On the SRA webpage, Click Select preload folder

Click Refresh folders

If some files were not transferred…

You can select specific files to transfer by using

put filename

Once all errors are corrected and files uploaded and they match your metadata, submission will go through a day of processing and you’ll get a confirmation email with your PRJ ID to include in publication.