# Hands-on : Local alignment and HIV drug resistance

## Background

### HIV lifecycle 
HIV is a virus that attacks the immune system. The figure below shows the life cycle of HIV.
```{figure} ./images/HIV_lifecycle.png
---
width: 400px
name: HIV_lifecycle
---
Lifecycle of HIV\
Source:https://hivinfo.nih.gov/understanding-hiv/fact-sheets/hiv-life-cycle
```

Note that **reverse transcription** is a key part of the life cycle.
Reverse transcription is carried out by an enzyme called **reverse transcriptase**, which is encoded in the HIV genome (segment labeled RT in the the pol region in the figure below of the structure of an HIV genome).
```{figure} ./images/HIV_genome.png
---
width: 600px
name: HIV_genome
---
Genome of HIV\
Source:https://hivinfo.nih.gov/understanding-hiv/fact-sheets/hiv-life-cycle
```

### Antiretroviral therapy
Antiretroviral therapy (ART) uses several kinds of drugs to keep HIV from multiplying. One class of ART drugs is called NRTI (Nucleoside Reverse Transcriptase Inhibitors), and as the name suggest, these interfere with the functioning of reverse transcriptase. The first drug approved for HIV ART use, Zidovudine (also called azidothymidine), falls in the NRTI class.

### Mutations and drug resistance 
Mutations in the RT gene can lead to altered reverse transcriptase enzymes, which are no longer affected by the drugs. [Here](https://www.iasusa.org/wp-content/uploads/2025/03/33-2-mutations.pdf) is a catalog of drug resistant mutations including those conferring resistance to NRTIs. [Here](https://hivdb.stanford.edu/cgi-bin/PositionPhenoSummary.cgi) is another one. 

What is worrisome is that we are seeing increasing drug resistance levels even in ART-naive patients, that is those have not initiated ART. 

### High-throughput sequencers for HIV surveillance
In recent years, high-throughput sequencers have been replacing the older Sanger sequencing technology in sequencing HIV genomes from patients to monitor drug resistance mutations. The newer sequencers can read a lot of sequences enabling discovery of variants that are present in low abundances.

[Here](http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0164156) is one study that uses modern sequencers to  survey the levels of HIV drug resistance in ART-naive individuals. The sequence data they produced is available [here](https://ddbj.nig.ac.jp/search/entry/sra-study/SRP083085).

## Objective
Identify and estimate the prevalence of HIV drug resistance mutations in an ART-naive individual by aligning sequence reads of HIV genome obtained from a patient to the reference HIV genome HXB2.

## Method
- Get HIV sequence data from 1 patient from the study above. This will be in [fastq](https://en.wikipedia.org/wiki/FASTQ_format) format. 
- Obtain the HXB2 reference sequence and annotation from [here](https://www.ncbi.nlm.nih.gov/nuccore/K03455.1).
- You will be using [LAST](https://gitlab.com/mcfrith/last) for the local alignment of reads to the reference.
    - Compute a suffix array of the reference using `lastdb`.
    - Train the scoring scheme using `last-train`.
    - Perform local alignment of reads to the reference using `lastal`.
    - Write a script to go through the sites of interest relevant to NRTI resistance (to make things simpler, you can just choose one drug e.g. zidovudine), and report on the mutations you can identify. 

## References
- John Herron, Scott Freeman, Chapter 1: A Case for Evolutionary Thinking: Understanding HIV, Evolutionary Analysis, Fifth Edition (2015), Pearson.
- https://www.iasusa.org/wp-content/uploads/2025/03/33-2-mutations.pdf
- https://hivdb.stanford.edu/cgi-bin/PositionPhenoSummary.cgi