# Submitting data to the NCBI Sequence Read Archive (SRA)


# Submitting data to the NCBI Sequence Read Archive (SRA)

## Pre-processing steps

- [ ] Collect fastq.gz files for each sample
- [ ] Rename fastq.gz files from long name to short name per sample
- [ ] Get biosample data early (especially if it's from collaborators)

## Links

- <https://submit.ncbi.nlm.nih.gov/subs/sra>
- <https://www.ncbi.nlm.nih.gov/sra/docs/submitfiles>
- [Sample attributes page](https://submit.ncbi.nlm.nih.gov/biosample/template/?package-0=MIMS.me.host-associated.6.0&action=definition)
- [Biosample batch page](https://www.ncbi.nlm.nih.gov/biosample/docs/submission/batch/)

## Basic Checklist

- [ ] Project Title 
- [ ] Public Description for Project 
- [ ] Grant Funding

## Start a New Submission

The example that I will provide is for a host-associated 16S sequencing study.

Go to the link above and start a submission. Fill out the project title, description, grant funding, and departmental information as requested.

Choose `Packages for metagenome submitters` if you are following along with host-associated 16S data, then `MIMS Environmental/Metagenome` from the `GSC MIxS` section on the right. Otherwise, fill out the form with your organism and follow along.

Choose `Upload a file using Excel or text format (tab-delimited) that includes the attributes for each of your BioSamples` and download the template.

## Minimum Biosample Checklist

**Note:** columns marked with `delete` should be completely deleted

Columns marked with `*` are required

`env_broad_scale` and `env_local_scale` will have a value if from environmental sources, but `not applicable` from host sources.

- [ ] *sample_name
- [X] sample_title - `delete`
- [X] bioproject_accession - `delete`
- [x] *organism - `mouse metagenome` (or other [host metagenome](https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Tree&id=410656&lvl=3&keep=1&srchmode=1&unlock))
- [ ] *collection_date - `YYYY-mm-dd` as one acceptable format
- [X] *env_broad_scale - `not applicable`
- [X] *env_local_scale - `animal-associated environment [ENVO:01001002]` (or other [host-environment](http://purl.obolibrary.org/obo/ENVO_01001002))
- [X] *env_medium - `fecal material [ENVO:00002003]` (or other [host tissue](http://purl.obolibrary.org/obo/ENVO_01000254))
- [X] *geo_loc_name - `USA: Oregon`
- [X] *host - `Mus musculus` (or other species binomial)
- [ ] *lat_lon - 44.566 N 123.283 W
- [ ] genetic_mod
- [X] host_common_name - `mouse` (or other species common name)
- [ ] host_diet
- [ ] host_genotype
- [ ] host_sex - `male` or `female`
- [ ] host_subject_id
- [ ] host_taxid - `10090` or other host taxid
- [ ] misc_param
- [ ] perturbation - experimental or control group
- [ ] neg_cont_type - `kit` or `water`

## Minimum SRA metadata

- [ ] sample_name - must match biosample name submitted above
- [ ] library_ID - can match sample_name
- [ ] title - `16S metabarcoding of Mus musculus: feces`
- [X] library_strategy - `AMPLICON`
- [X] library_source - `METAGENOMIC`
- [X] library_selection - `PCR`
- [X] library_layout - `paired`
- [X] platform - `ILLUMINA`
- [X] instrument_model - `Illumina MiSeq`
- [ ] design_description - choose from below
  - `Earth Microbiome Project 16S PCR protocol`
  - `Illumina 16S PCR protocol`
- [X] filetype - `fastq`
- [ ] filename - `sample_name_R1.fastq.gz`
- [ ] filename2 - `sample_name_R2.fastq.gz`
- [ ] filename3	- `delete`
- [ ] filename4 - `delete`
- [ ] assembly - `delete`
- [ ] fasta_file - `delete`

## Uploading data using ftp

- Expand the FTP instructions
- Log in to files.cqls.oregonstate.edu using ssh
- Navigate to the directory containing the reads
- Connect to NCBI over ftp using given credentials:
  - `ftp -i ftp-private.ncbi.nlm.nih.gov`
  - `-i` flag allows multiple transfers without confirming
- Change to your given directory on the website
- `mkdir` a new directory for this submission
- Use `mput` to upload multiple files at once
  - `mput *.fastq.gz`
- Wait until the files are available to select in the web interface
  - May take **10+ minutes**
- Choose the direcory in the `Select preload folder` dialog and click continue