Introduction to SRA Aligned Read Format and Cloud Metadata for SARS-CoV-2

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# Introduction to SRA Aligned Read Format and Cloud Metadata for SARS-CoV-2

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		Authors: 
<a href="/training-material/hall-of-fame/jontrow/" class="contributor-badge contributor-jontrow"><img src="https://avatars.githubusercontent.com/jontrow?s=27" alt="Avatar">Jon Trow</a>

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<div class="footnote" style="bottom: 8em;"><i class="far fa-calendar" aria-hidden="true"></i><span class="visually-hidden">last_modification</span> Updated: Jun 2, 2021</div>

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### <i class="far fa-question-circle" aria-hidden="true"></i><span class="visually-hidden">question</span> Questions

- What is the SRA Aligned Read Format

- How can I use it?

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### <i class="fas fa-bullseye" aria-hidden="true"></i><span class="visually-hidden">objectives</span> Objectives

- Understand which data and metadata types are available for SARS-CoV-2 SRA runs in the cloud.

- Understand what SRA Aligned Read Format is and what can you do with it.

- Working knowledge of SRA Metadata available on the cloud, ways to access it, and some popular use cases.

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# Why have SRA data in the cloud?

- SRA data archive format is over 18PB (**great!**)

...but it's a lot of data that is hard to search (**not so great**)

- Do you wish:
  - It was easier to find SRA data based on organismal content?
  - Searching SRA based on submitted BioProject and BioSample information was simpler?
  - You could get a sense of what could be assembled out of an SRA dataset before downloading?
  - You didn't need to do the assembly/variant calling yourself?

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## What data is in scope for this training?

- Public SRA data that contains SARS-CoV-2 sequence
	- Illumina platform only
	- Stay tuned for long-read data

- How do we determine which runs contain SARS-CoV-2 data?
	- SRA Taxonomy Analysis Tool (STAT)
	- Preprint: [doi:10.1101/2021.02.16.431451v1](https://www.biorxiv.org/content/10.1101/2021.02.16.431451v1)
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## Introduction to SRA aligned read format and associated metadata

- Incoming SRA runs are scanned for *Coronaviridae* content
- Contigs are created via guided assembly against SARS-CoV-2 RefSeq - fasta contigs
- Reads are mapped back to the contigs - aligned sam, fasta/q reads
- Taxonomy assessed
	- STAT
	- BLAST
- Variants are called then annotated with VIGOR3 - VCF

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## Using aligned read format

.pull-left[
- *Coronaviridae* contigs constructed from SRA data
	- Compatible with SRA Toolkit
	- Access to reads and contigs individually
	- Ability to see which reads align to each contig
	- Smaller size than standard SRA format- no quality scores
]

.pull-right[
![graph with one large node of SRA aligned reads leading to three other nodes: FASTQ, SAM, Fasta contigs (new)](../../images/ncbi/sarf.png)
]

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## Accessing SRA metadata in the cloud

- Cloud query services- BigQuery and Athena
	- Analyze SRA metadata directly using standard SQL, allowing searches that are not possible in Entrez
	- No need to manually transform the data for querying, so you can be up and running in just a few minutes
	- Built in support for complex data fields
	- You pay for the data scanned, though some free options exist
- The underlying metadata files are available for download at no cost
	- JSON format can be transformed according to your preferences
	- These can be imported directly to Galaxy

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## Available SRA metadata

SRA aligned read metadata:

- User submitted metadata
	- Biological sample and sequencing library metadata supplied at the time of sequence submission
- Contig metadata
	- Contig stats including: coverage, taxonomy ID, contig length
- Taxonomic content analysis of contigs
	- Search for records based on kmer hits to tax level of interest
- BLAST results for contigs
	- Contig blast results including: hit accession, hit length, percent identity
- Variant calls
	- Variation and VIGOR3 annotation information
- Peptides
	- Details of annotated peptides, including sequence

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## Popular use cases- what can you do with it?

- Finding SARS-CoV-2 contigs with specific coverage and length
- Filter SARS-CoV-2 contigs based on geographic location of sample collection
- Find top SARS-CoV-2 BLAST hits for contigs from a specific geographic location
- Filter for all variations called from samples collected in the United States after 02/01/2021
- Filter for runs with the E484K mutation

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## SRA data in the cloud

.pull-left[
- COVID-focused SRA datasets available in GCP and AWS
	- Searchable metadata
	- SRA aligned read files
	- VCF files
	- Full SRA runs
	- Original format submitted files
- Benefits
	- Access large volume of COVID-19-related raw read data with no throttling, at no cost to you
	- Can be imported into Galaxy for use in your existing workflows
]

.pull-right[
.image-100[![screenshot of google cloud ncbi covid-19 project website](../../images/ncbi/covid-cloud.png)]
.image-100[![screenshot of aws ncbi covid-19 project website](../../images/ncbi/covid-aws.png)]
]

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### <i class="fas fa-key" aria-hidden="true"></i><span class="visually-hidden">keypoints</span> Key points

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## Thank You!

This material is the result of a collaborative work. Thanks to the [Galaxy Training Network](https://training.galaxyproject.org) and all the contributors!

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<a rel="license" href="https://creativecommons.org/licenses/by/4.0/">
This material is licensed under the Creative Commons Attribution 4.0 International License</a>.