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
• Biosample batch page

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
• sample_title - delete
• bioproject_accession - delete
• *organism - mouse metagenome (or other host metagenome)
• *collection_date - YYYY-mm-dd as one acceptable format
• *env_broad_scale - not applicable
• *env_local_scale - animal-associated environment [ENVO:01001002] (or other host-environment)
• *env_medium - fecal material [ENVO:00002003] (or other host tissue)
• *geo_loc_name - USA: Oregon
• *host - Mus musculus (or other species binomial)
• *lat_lon - 44.566 N 123.283 W
• genetic_mod
• 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
• library_strategy - AMPLICON
• library_source - METAGENOMIC
• library_selection - PCR
• library_layout - paired
• platform - ILLUMINA
• instrument_model - Illumina MiSeq
• design_description - choose from below
  • Earth Microbiome Project 16S PCR protocol
  • Illumina 16S PCR protocol
• 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