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exercise-01_16s.qmd
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exercise-01_16s.qmd
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# 16S Sequence Taxonomic Identification
## Introduction
In this exercise you will use the 16S sequence that you amplified from your unknown bacterium in order to make a taxonomic identification.
To do this, you will use two online bioinformatics services:
1. NCBI's [`BLAST` tool](https://blast.ncbi.nlm.nih.gov/Blast.cgi) (you have already met this in [Workshop 2](https://sipbs-compbiol.github.io/BM214-Workshop-2/scenario-1.html))
2. [`silva`](https://www.arb-silva.de/), a quality-checked and curated 16S sequence database service
{#fig-biokittenformatics width=60%}
::: { .callout-caution collapse="true"}
## Why are we using two different services?
It might tempting to take computational results at face value, and to believe that the "computer can't be wrong." But, _in bioinformatics as elsewhere in life_, **computers and databases can sometimes be in error or give imprecise answers**. In general the results of a sequence database search, like querying a 16S sequence against a public database, depend on a number of factors, including:
- the query sequence (obviously)
- the contents of the database
- the search tool used to query the database
- the parameter settings for the search tool
and even if the query sequence is the same, using a different service will often change at least one of the other three factors - and maybe other thigs besides.
**This exercise will give you experience in using alternative bioinformatics tools for the same task.**
:::
## Your data
::: { .callout-important }
**You should download the 16S sequence file you obtained from your experiment in Laboratory 3.**
This will be a plain-text file in [FASTA format](glossary.qmd#fasta-format).
:::
## Analysis 1: NCBI `BLAST`
Follow the steps below to carry out a [`blastn`](glossary.qmd#blastn) search using your 16S sequence data.
### Navigate to the NCBI `BLAST` webservice
- NCBI [`BLAST` homepage](https://blast.ncbi.nlm.nih.gov/Blast.cgi)
### Select the BLAST Tool
::: { .callout-tip collapse="true"}
## Hint: Which `BLAST` tool should I use?
Your query sequence is a nucleotide sequence, and the 16S sequences of other organisms are also nucleotide sequences, so this is a **nucleotide vs nucleotide** search.
For a reminder, see [Workshop 2](https://sipbs-compbiol.github.io/BM214-Workshop-2/blast.html).
:::
::: { .callout-warning collapse="true"}
## Answer: The `BLAST` tool to use
This is a **nucleotide vs nucleotide** search, so use the `Nucleotide BLAST` tool.
{#fig-nucleotide-blast-cover width="80%"}
:::
### Enter the query sequence
::: { .callout-warning collapse="true"}
## Answer: Entering the query sequence
**You can enter the query sequence in either of two ways.**
1. Open your 16S sequence file in a text editor, copy the data, and paste the data into the `Enter Query Sequence` field.
{#fig-scenario-1-query width="80%"}
2. Click on the `Browse…` button to open a file selection dialogue box, then select your 16S sequence file. The uploaded file's name will then be visible next to the `Browse…` button.
{#fig-blastn-filename width="80%"}
:::
### Set appropriate parameter choices
::: { .callout-tip collapse="true"}
## Hint: Which database should I use?
The NCBI `BLAST` webservice provides specialised databases for common, well-defined search tasks.
You have a well-defined set of target sequences that you want to compare your query with: **they are all 16S sequences.** Can you see a database on the search page that might be suitable?
:::
::: { .callout-warning collapse="true"}
## Answer: Which database to use
The NCBI `BLAST` webservice provides specialised databases for common, well-defined search tasks, including searching for 16S or ITS (Internally-Transcribed Spacer) sequences when identifying unknown microorganisms.
**Select the `rRNA/ITS databases` radio button, and ensure that the "Bacteria and Archaea" database is selected.**
{#fig-scenario-1-database width="80%"}
:::
### Run the `BLAST` search
::: { .callout-warning collapse="true"}
## Answer: Running the `BLAST` search
Click on the `BLAST` button.
{#fig-scenario-1-button-1 width="80%"}
:::
### Interpret the `BLAST` report (**MyPlace Questions**)
It is common for 16S rDNA sequences to match many other bacterial sequences in the database. However, by identifying those most closely-related to the query sequence, you should be able to establish a likely identity for this organism.
::: { .callout-important title="Discussion Questions"}
**As you all have different sequences there is no MyPlace quiz, please answer these questions for yourself.**
_If you would like guidance about your answer, please ask one of the tutors in the workshop._
Clicking on the green box should give you a hint to the answer, or where to find it.
:::
::: { .callout-tip collapse="true"}
## What is the accession number of the best match to your query?
Check the `Accession` column under "Sequences producing significant alignments" in the report's `Descriptions` tab.
:::
::: { .callout-tip collapse="true"}
## What organism is the best match to your query?
Check the `Description` column under "Sequences producing significant alignments" in the report's `Descriptions` tab.
:::
::: { .callout-tip collapse="true"}
## What is the total score for the best matching alignment?
Check the `Total Score` column under "Sequences producing significant alignments" in the report's `Descriptions` tab.
:::
::: { .callout-tip collapse="true"}
## What is the query coverage against the best matching alignment?
Check the `Query Cover` column under "Sequences producing significant alignments" in the report's `Descriptions` tab.
:::
::: { .callout-tip collapse="true"}
## What is the alignment percentage identity for the best matching alignment?
Check the `Per. Ident` column under "Sequences producing significant alignments" in the report's `Descriptions` tab.
:::
## Analysis 2: `silva`
[`silva`](https://www.arb-silva.de/) is a curated, quality-checked database of rRNA sequence data that has been run by the [Liebniz Institute DSMZ German Collection of Microorganisms and Cell Cultures](https://www.dsmz.de/) for about two decades. The `silva` site provides a number of online tools and services, including the [ACT](https://www.arb-silva.de/aligner) service that enables users to search the database with their own rRNA sequences to find the best matches and identify their organism.
- [`silva` rRNA database project](https://www.arb-silva.de/)
- [`silva` Alignment, Classification, and Tree (ACT) webservice](https://www.arb-silva.de/aligner)
- [ACT online tutorial](https://www.arb-silva.de/documentation/act-tutorial/)
### Navigate to the `silva` webservice
- [`silva` ACT](https://www.arb-silva.de/aligner) (Alignment, Classification, and Tree) service
{#fig-silva-01 width=80%}
### Enter the query sequence
::: { .callout-warning collapse="true"}
## Answer: Entering the query sequence
**You can enter the query sequence in either of two ways.**
1. Open your 16S sequence file in a text editor, copy the data, and paste the data into the `Input data` field (where it says "Paste your FASTA sequence here").
{#fig-silva-02 width=80%}
2. Click on the `Select file` button to open a file selection dialogue box, then select your 16S sequence file. The uploaded file's name will then be visible in the field next to the `Select file` button.
{#fig-silva-03 width=80%}
:::
### Set appropriate parameter choices
::: { .callout-tip collapse="true" }
## Hint: What parameters are important?
`silva` is designed specifically to assign taxonomy from 16S sequence data, so you shouldn't usually need to modify any parameters. However, you do need to tell `silva` what actions you want it to take.
You want to **search** with your sequence **and classify** it.
:::
::: { .callout-warning collapse="true" }
## Answer: What parameters to set
You need to tell `silva` that you want to **search** with your sequence **and classify** it. So **check the box marked `Search and classify`.**
The default settings (95% minimum sequence identity, reporting ten neighbours) are a good starting point.
Once you select `Search and classify`, a new set of options will appear in the window (@fig-silva-04) and these can also be left as they are.
{#fig-silva-04 width=80%}
:::
### Run the `silva` search
::: { .callout-warning collapse="true" }
## Answer: Running the `silva` search
Click on the `Run Tool` button (lower right of @fig-silva-04).
:::
::: { .callout-caution }
**`silva` does not provide results as quickly as NCBI's `BLAST` server. Keep your eye on the `Aligner Taskmanager` section of the page, to monitor progress.**
`silva`'s progress will run through several stages, from `initialising` to `finished`.
{#fig-silva-05 width=80%}
:::
### Interpret the `silva` report (**MyPlace Questions**)
Unlike `BLAST`, `silva` provides the identity of the [_last common ancestor_ (LCA)](https://evolution.berkeley.edu/glossary/most-recent-common-ancestor/), rather than reporting a sequence search match. It displays results in the `Alignment Result Table` (@fig-silva-06). This table displays scores but, by clicking on the `Display Classification` button, you can see the taxonomic identification `silva` has made for your sequence (@fig-silva-07).
{#fig-silva-06 width=80%}
{#fig-silva-07 width=80%}
::: { .callout-tip collapse="true" }
## Help! I can't see the full classification!
Use the "handles" to drag the size of the column headers to the left or right, until you can see the classification, as in @fig-silva-08.
{#fig-silva-08 width=80%}
Alternatively, use the `Export to CSV` option to download a plain-text `.csv` file containing `silva`'s results for your sequence. You can open this in a text editor, or in Excel.
:::
::: { .callout-important title="Discussion Questions"}
**As you all have different sequences there is no MyPlace quiz, please answer these questions for yourself.**
_If you would like guidance about your answer, please ask one of the tutors in the workshop._
Clicking on the green box should give you a hint to the answer, or where to find it.
:::
::: { .callout-tip collapse="true"}
## What is the the taxonomic identity of the last common ancestor (LCA) `silva` assigns to your organism?
You can find this in the `LCA tax. SILVA` column of the `Alignment Result Table`, or in the `lca_tax_slv` column of the downloaded `.csv` file.
:::
::: { .callout-tip collapse="true"}
## What sequence identity does your isolate's sequence share with the sequence of the reported last common ancestor?
You can find this in the `Identity` column of the `Alignment Result Table`, or in the `identity` column of the downloaded `.csv` file.
:::
::: { .callout-tip collapse="true"}
## Do the `BLAST` and `silva` searches give the same taxonomic identity for your 16S sequence?
Inspect the outputs of both methods, and compare the taxonomic assignments.
:::
::: { .callout-tip collapse="true"}
## What is your current opinion about the identity of your isolate? How confident are you in the identification made by each tool?
Among other things, you may want to consider:
- did both tools give the same identification?
- how much sequence identity was shared between your query sequence and the matches each tool found?
- is your organism common enough that there are many examples of its 16S sequence known?
:::
## Summary
::: { .callout-note title="Well Done!"}
After successfully working through this section you should be able to:
- use `BLAST` and `silva` to obtain taxonomic identity from 16S sequence data
- interpret `BLAST` and `SILVA` output
- explain the difference in results between `BLAST` and `silva` taxonomy assignment
:::