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### Genomic data standards

---

## What is a standard?

> A norm or specification applied to a repeated process. It includes a formal definition of terms and a description of formats and expected behavior of a system

<p class="fragment">Standards → Interoperability</p>
---

What happens when there is no standard  
(or, equivalently, too many standards)

---

---

### Existing standards in genomics

- Project metadata
- Ontologies
- File formats
- Reference genomes
- Web standards and APIs

<p>Standards → Interoperability</p>

---
### GA4GH

> The Global Alliance for Genomics and Health (GA4GH) is a policy-framing and technical standards-setting organization, seeking to enable responsible genomic data sharing within a human rights framework.

- [ga4gh.org](https://www.ga4gh.org/)
- formed in 2013

---

<h3>Research is organized in <i>projects</i></h3>
<div class="row">
<div class="col">
<img src="images/genomic-data-standards/tubes.svg" width="450">
</div>
<div class="col fragment">
<img src="images/genomic-data-standards/tubes_color.svg" width="450">
</div>
</div>
<div class="fragment">
  How do we conceptualize a research project?
</div>

---

<h2>Each project has 3 components</h2>
<img src="images/genomic-data-standards/data-code-compute.svg" width="500" style="align:center"><br/>

---

<h2>Organizing multiple projects is a challenge</h2>
<img src="images/genomic-data-standards/linking-projects.svg" width="600" style="align:center"><br/>

---

<h2>How do I re-use a component?</h2>
<img src="images/genomic-data-standards/linking-projects-reuse.svg" width="600" style="align:center"><br/>

---

<h2>A project is a set of edges in a tripartite graph</h2>
<img src="images/genomic-data-standards/linking-projects-modular.svg" width="600" style="align:center"><br/><br/>

---

<h2>Enable linking with interfaces</h2>
<img src="images/genomic-data-standards/linking-interfaces.svg" width="600" style="align:center"><br/>

---

### Existing standards in genomics

- Project metadata (Data → Code)
- Ontologies (Data → Code)
- File formats (Data → Data)
- Reference genomes (Data → Data)
- Web standards and APIs (Data → Compute)

<p>Standards → Interoperability</p>

---

<h2>PEP: Portable Encapsulated Projects</h2>
<img src="images/genomic-data-standards/pep_center_white.svg" width="700">
---
<img src="images/genomic-data-standards/pep_contents_white.svg" width="750">
---
<div class="bullet">
  <h2><img src="images/genomic-data-standards/pep_logo.svg" width="70">PEP format</h2>
</div>
<div class="bullet">
<img src="images/genomic-data-standards/file.svg" width="30">project_config.yaml
</div>
<pre><code>sample_table: /path/to/samples.tsv
output_dir: /path/to/output/folder
</code></pre>
<hr>
<div class="bullet">
<img src="images/genomic-data-standards/file.svg" width="30">samples.csv
</div>
<pre><code>sample_name, protocol, organism, data_source
frog_0h, RNA-seq, frog, /path/to/frog0.gz
frog_1h, RNA-seq, frog, /path/to/frog1.gz
frog_2h, RNA-seq, frog, /path/to/frog2.gz
frog_3h, RNA-seq, frog, /path/to/frog3.gz
</code></pre>

---

<h3>PEP portability features</h3>
  <div style="text-align: left">

    <span class="bullet"><img src="images/genomic-data-standards/replace_white.svg" width="50" class="bullet">Derived attributes</span><br>
    <span class="bullet"><img src="images/genomic-data-standards/implies_white.svg" width="50" class="bullet">Implied attributes</span><br>
    <span class="bullet"><img src="images/genomic-data-standards/subproject_white.svg" width="50" class="bullet">Subprojects</span><br>
  </div>

---

<span class="bullet"><img src="images/genomic-data-standards/replace_white.svg" width="50" class="bullet">Derived attributes</span><br>
<div class="well">Build new sample attributes from existing ones</div>

Without derived attribute:
<code>
<div data-markdown style="font-size:0.4em">

| sample_name   | t    | protocol        | organism | data_source            |
| ------------- | ---- | :-------------: | -------- | ---------------------- |
| frog_0h       | 0    | RNA-seq         | frog     | /path/to/frog0.gz      |
| frog_1h       | 1    | RNA-seq         | frog     | /path/to/frog1.gz      |
| frog_2h       | 2    | RNA-seq         | frog     | /path/to/frog2.gz      |
| frog_3h       | 3    | RNA-seq         | frog     | /path/to/frog3.gz      |

</div>
</code>

Using derived attribute:
<code>
<div data-markdown style="font-size:0.4em">

| sample_name   | t    | protocol        | organism | data_source            |
| ------------- | ---- | :-------------: | -------- | ---------------------- |
| frog_0h       | 0    | RNA-seq         | frog     | my_samples             |
| frog_1h       | 1    | RNA-seq         | frog     | my_samples             |
| frog_2h       | 2    | RNA-seq         | frog     | my_samples             |
| frog_3h       | 3    | RNA-seq         | frog     | my_samples             |
| crab_0h       | 0    | RNA-seq         | crab     | your_samples           |
| crab_3h       | 3    | RNA-seq         | crab     | your_samples           |

</div>
</code>
---
<code>
<div data-markdown style="font-size:0.5em">

</div>
</code>
Project config file:

```yaml
sample_modifiers:
  derive:
    attributes: [data_source]
    sources:
      my_samples: "/path/to/my/samples/{organism}_{t}h.gz"
      your_samples: "/your/samples/{organism}_{t}h.gz"
```

{variable} identifies sample annotation columns

<div class="well">Benefit: Enables distributed files, portability</div>

---

Implied attributes

<div class="well">Add new sample attributes conditioned on values of existing attributes</div>
<div class="col2">
Before:<br>
<code>
<div data-markdown style="font-size:0.5em">

| sample_name   | protocol        | organism | 
| ------------- | :-------------: | -------- | 
| human_1       | RNA-seq         | human    | 
| human_2       | RNA-seq         | human    | 
| human_3       | RNA-seq         | human    | 
| mouse_1       | RNA-seq         | mouse    |

</div>
</code>
</div>

<div class="col2">
After:<br>
<code>
<div data-markdown style="font-size:0.5em">

| sample_name   | protocol        | organism | genome | 
| ------------- | :-------------: | -------- | ------ |
| human_1       | RNA-seq         | human    | hg38   |
| human_2       | RNA-seq         | human    | hg38   |
| human_3       | RNA-seq         | human    | hg38   |
| mouse_1       | RNA-seq         | mouse    | mm10   |

</div>
</code>
</div>
---
<code>
<div data-markdown style="font-size:0.5em">

</div>
</code>
Project config file:

```yaml
sample_modifiers:
  imply:
    - if: 
        organism: human
      then:
        genome: hg38
    - if: 
        organism: mouse
      then:
        genome: mm10
```

<div class="well">Benefit: Divides project from sample metadata</div>

---
Project amendments

<div class="well">Define activatable project attributes.</div>

```yaml
project_modifiers:
  amend:
    diverse:
      sample_table: psa_rrbs_diverse.csv
    cancer:
      sample_table: psa_rrbs_intracancer.csv
```

<div class="well">Benefit: Defines multiple similar projects in a single file</div>

---
### Is it enough?

Study 1:
<code>
<div data-markdown style="font-size:0.4em">

| sample_name   | t    | protocol        | organism | data_source            |
| ------------- | ---- | :-------------: | -------- | ---------------------- |
| frog_2h       | 2    | RNA-seq         | frog     | my_samples             |
| frog_3h       | 3    | RNA-seq         | frog     | my_samples  
</div>
</code>

Study 2:
<code>
<div data-markdown style="font-size:0.4em">

| sample_name   | t    | protocol        | organism | data_source            |
| ------------- | ---- | :-------------: | -------- | ---------------------- |
| frog_0m       | 0    | RNAseq         | frog     | my_samples             |
| frog_60m       | 60  | RNAseq         | frog     | my_samples             |
</div>
</code>

---

### Ontologies

> A formal vocabulary and definition of concepts, entities, and their relationships.

1. Terms
2. Relations

---

## Gene Ontology

- 3 Ontologies:
  - Molecular Function
  - Cellular Component
  - Biological Process

1. Terms: [Hexose Biosynthetic Process](http://amigo.geneontology.org/amigo/term/GO:0019319)

2. Relations: is-a,  is-part-of, regulates ([Relation Ontology](https://doi.org/10.1186/gb-2005-6-5-r46))

---

![](images/genomic-data-standards/hexose-biosynthetic-process.png)

---
### EDAM Ontology

![](images/genomic-data-standards/edam-ontology.png)

---
### Sequence Ontology

Term: `open_chromatin_region`  
SO Accession: `SO:0001747`

![](images/genomic-data-standards/SO0001747_sm.png)

</div>
<div class="col">

Definition:  
A DNA sequence that in the normal state of the chromosome corresponds to an unfolded, un-complexed stretch of double-stranded DNA.

</div>
</div>

---
<img src="images/genomic-data-standards/SO0000162_lg.png" height="670">

---
How can we enforce or validate terms?

Study 1:
<code>
<div data-markdown style="font-size:0.4em">

Study 2:
<code>
<div data-markdown style="font-size:0.4em">

---
### JSON Schema

```
{
  "productId": 1,
  "productName": "A green door",
  "price": 12.50,
  "tags": [ "home", "green" ]
}
```

- What is productId?
- Is productName required?
- Can the price be zero (0)?
- Are all of the tags string values?

---
### RNA-seq analysis

```
{
  "sample_name": frog_2h,
  "t": "2",
  "protocol": "RNA-seq",
  "organism": "frog",
  "data_source": "my_samples"
}
```

- Is sample_name required?
- What is t?
- What are the allowable protocols?
- What are the allowable organisms?
- What is the input file type expected for data source?

---
### Schema

```
{
  "title": "Product",
  "description": "A product from Acme's catalog",
  "type": "object",
  "properties": {
    "productId": {
      "description": "The unique identifier for a product",
      "type": "integer"
    },
    "productName": {
      "description": "Name of the product",
      "type": "string"
    }
  },
  "required": [ "productId", "productName" ]
}
```
---
### Validating objects with schemas

```shell
jsonschema --instance sample.json sample.schema
```

```python
validate([2, 3, 4], {"maxItems": 2})
Traceback (most recent call last):
    ...
ValidationError: [2, 3, 4] is too long
```
---
### What is a schema?

A formal definition of the properties and allowable values for data

<span class="fragment">
JSON-Schema provides a standard format for how to describe generic data objects, and tools to validate them.
</span>

---
### A schema for ATAC-seq pipeline

```
description: A PEP for ATAC-seq samples for the PEPATAC pipeline.
imports:
  - http://schema.databio.org/pep/2.0.0.yaml
properties:
  samples:
    type: array
    items:
      type: object
      properties:
        sample_name: 
          type: string
          description: "Name of the sample"
        organism: 
          type: string
          description: "Organism"
        protocol: 
          type: string
          description: "Must be an ATAC-seq or DNAse-seq sample"
        genome:
          type: string
          description: "Refgenie genome registry identifier"
        read_type:
          type: string
          description: "Is this single or paired-end data?"
          enum: ["SINGLE", "PAIRED"]
        read1:
          anyOf:
            - type: string
              description: "Fastq file for read 1"
            - type: array
              items:
                type: string
        read2:
          anyOf:
            - type: string
              description: "Fastq file for read 2 (for paired-end experiments)"
            - type: array
              items:
                type: string
      required_files:
        - read1
      files:
        - read1
        - read2
      required:
        - sample_name
        - protocol
        - read1
        - genome
required:
  - samples
```

---

---

### Standards: File formats
  - FASTA: DNA sequences
  - FASTQ: Short DNA sequencer reads
  - SAM/BAM: Aligned DNA sequences
  - BED: genomic intervals
  - VCF: genomic variation

---
### File formats are not enough

Say I have two BED files. Are they comparable?

File 1: 
```
chr1  15300  17300
chr1  24900  25600
chr1  72420  74440
```

File 2:
```
1  18110  19300
1  64000  65600
1  72400  74700
```

<div class="fragment">sequence names must match</div>

<div style="color:black">.</div>
---
### File formats are not enough

Say I have two BED files. Are they comparable?

File 1: 
```
chr1  15300  17300
chr1  24900  25600
chr1  72420  74440
```

File 2:
```
chr1  18110  19300
chr1  64000  65600
chr1  72400  74700
```

sequence names must match. How about now?

<div class="fragment">reference sequence must be compatible</div>
---
<div class="row" style="border: 1px solid white">
	<div class="col" style="border: 1px solid white"></div>
	<div class="col" style="border: 1px solid white">Names Match</div>
	<div class="col" style="border: 1px solid white">Names Mismatch</div>
</div>
<div class="row" style="border: 1px solid white">
	<div class="col" style="border: 1px solid white">Reference Match</div>
	<div class="col" style="border: 1px solid white">Great!</div>
	<div class="col" style="border: 1px solid white">Adjust names?</div>
</div>
<div class="row" style="border: 1px solid white">
	<div class="col" style="border: 1px solid white">Ref. Mismatch</div>
	<div class="col" style="border: 1px solid white">Don't be fooled!</div>
	<div class="col" style="border: 1px solid white">No compatibility</div>
</div>
---
### Standards: Reference genomes

Reference genomes are versioned.

human reference: hg18, hg19, hg38/GRCh38, T2T

mouse reference: mm8, mm9, mm10

They are not compatible!

---
### Converting among genome builds

Liftover ([Luu et al. 2020](https://doi.org/10.1093/nargab/lqaa054))

---
### Standards: Reference genomes

There are also many variations of the *same* version.

- 3 GRCh38 providers: NCBI, Ensembl, UCSC
- hard- soft-, or no repeat masks?
- what are the chromosomes named (1 vs chr1 vs chr1, NC_000001.11)
- what secondary scaffolds are included?
- how is the assembly named (hg38, GRCh38, or GCF_000001405.39)?
- are any decoy sequences included (like EBV)?

<div class="fragment">
Downstream results are not compatible if based on a different reference!
</div>

---
Andy Yates' "Genome provider analysis"
<table class="tg" style="margin-bottom:60px">
<thead>
  <tr>
    <th class="tg">Provider</th>
    <th class="tg">Chr1 name</th>
    <th class="tg">Chr1 length</th>
    <th class="tg">Chr1 md5</th>
    <th class="tg">Num chroms</th>
  </tr>
</thead>
<tbody>
  <tr>
    <td class="tg">Ensembl primary</td>
    <td class="tg">1</td>
    <td class="tg">248956422</td>
    <td class="tg">2648ae1bacce4ec4b6cf337dcae37816</td>
    <td class="tg">195</td>
  </tr>
  <tr>
    <td class="tg">Ensembl toplevel</td>
    <td class="tg">1</td>
    <td class="tg">248956422</td>
    <td class="tg">2648ae1bacce4ec4b6cf337dcae37816</td>
    <td class="tg">649</td>
  </tr>
  <tr>
    <td class="tg">NCBI</td>
    <td class="tg">NC_000001.11</td>
    <td class="tg">248956422</td>
    <td class="tg">6aef897c3d6ff0c78aff06ac189178dd</td>
    <td class="tg">640</td>
  </tr>
  <tr>
    <td class="tg">UCSC</td>
    <td class="tg">chr1</td>
    <td class="tg">248956422</td>
    <td class="tg">2648ae1bacce4ec4b6cf337dcae37816</td>
    <td class="tg">456</td>
  </tr>
</tbody>
</table>

<div class="small">https://gist.github.com/andrewyatz/692f81baab1bebaf09c481937f2ad6c6</div>

---
### Hashing

> A hash function maps an object to a finite space

- must be deterministic
- should be very fast to compute
- should reduce collisions

<div class="fragment">Used for hash tables, but also...</div>

---
### Hashing

The output, called a *hash* or *digest*, can be seen as a fingerprint or barcode. It is a unique identifier of an input item.

<div class="fragment">As long as there are no collisions...</div>

---

### How [GA4GH:refget](https://doi.org/10.1093/bioinformatics/btab524) works

---
### Sanitize sequence before digesting

1. Remove whitespace
2. Make upper-case

---
### CRAM format

Can use refget server for references

---
Refget is great, but:

- what about *collections* of sequences, like a reference genome?
- what about *names* of sequences?

---

<h2>Sequence collections</h2>

Digest algorithm

---

<h2>Sequence collections</h2>

Digest lookup

---

hg38 → a6748aa0f

---

✓ File formats  
✓ Reference genome

Are these RNA-seq experiments comparable?

File 1: 
```
         sample1   sample2   sample3
MIR1302        7        2        153
FAM138A       64       30         55  
LINC01409     14       22         17  
PRDM16-DT      2        0          0  
```

File 2:
```
                  sample1   sample2   sample3
ENSG00000243485        64        22        55  
ENSG00000237613.2      17        53        52  
ENSG00000238009.6      12        93       455  
ENST00000477740.5       2        35         7  
```

---
### Existing standards in genomics
- Gene names
  - HUGO Gene Nomenclature Committee (https://www.genenames.org/)
- Gene and transcript annotation
  - GENCODE (https://www.gencodegenes.org/)

---
### RNA-seq

---
### Transcript models

![](images/genomic-intervals/brca2-gene-model.png)

---

---

- Web standards and APIs (Data → Compute)

---
### The increasing importance of web standards

As data and analysis become larger and more distributed, more recent standards increase involvement of web concepts

---
### Application programming interface (API)

- An API defines protocols and formats for input and output of a computing system
- It provides an abstraction layer to a user

---
### GA4GH:REFGET

[Refget API Specification](http://samtools.github.io/hts-specs/refget.html)

```python
GET /sequence/{id}
# get sequence by ID. The primary method 
# for accessing specified sequence data. 
```

---
### GA4GH:DRS

[Data Repository Service (DRS)](https://ga4gh.github.io/data-repository-service-schemas/preview/release/drs-1.0.0/docs/)

> Provides a generic interface to data repositories so data consumers, including workflow systems, can access data in a single, standard way regardless of where it’s stored and how it’s managed

```
GET /objects/{object_id}
# returns a JSON response including access methods 
```

```
/objects/{object_id}/access/{access_id}
# returns a URL where you can get the object  
```

---
### GA4GH:DRS

#### Current approach

You share your data with a link on your website

#### Future approach

You implement DRS. Then, a standard DRS client could get data, making your data more accessible.

</div>

---
### GA4GH:WES

[Workflow Execution Service](https://ga4gh.github.io/workflow-execution-service-schemas/docs/)

Provides a standard way for users to submit workflow requests to workflow execution systems, and to monitor their execution

- GET `/runs` - to list current runs
- POST `/runs` - to start a new run
- GET `/runs/{run_id}` - information about a run
- GET `/runs/{run_id}/status` - status of a run
- POST `/runs/{run_id}/cancel` - cancels a run

---
### GA4GH:TES

[Task Execution Service](https://ga4gh.github.io/task-execution-schemas/docs/)

- GET `/tasks` - list tasks tracked by the TES server
- POST `/tasks` - create new task
- GET `/tasks/{id}` - get a single task
- POST `/tasks/{id}:cancel` - cancel a task

---

### Conclusion

Who is the target audience?

- humans?
- computers?

<div class="fragment">It's easy to build a human-friendly interface to data organized for computers. The opposite is not true.</div>

<div class="fragment">
So, always build in this direction:

computers → humans

</div>

---

1. Make and share *everything* in *computable* form
  - your raw and processed data
  - your results
  - your writing (markdown)
  - computability precedes interoperability
2. Follow standard file formats
3. Use controlled terms (e.g. ontologies)
4. Identify elements with unique identifiers

---

- Genomes will always be a community resource
  - It's a huge, worldwide, rapidly growing endeavor
  - Be familiar with existing standards
  - Use them whenever possible
  - Be aware of work on new standards

<div class="fragment">
Remember, interoperability means impact
</div>