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armintoepfer committed Apr 11, 2021
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# PacBio Amplicon Analysis (_pbaa_)
<p align="center">
<img src="img/pbaa_logo_transparent.png" alt="pbaa logo" width="250px"/>
</p>
<h1 align="center"><i>pbaa</i></h1>
<p align="center">PacBio Amplicon Analysis</p>

***

PacBio Amplicon Analysis (_pbaa_) separates complex mixtures of amplicon targets from genomic samples. The _pbaa_ application is designed to cluster and generate high-quality consensus sequences from HiFi reads. This application only works on HiFi amplicon data. There are several assumptions made within the code that will only support high quality reads (>QV20). This application will not work on CLR data. _pbaa_ is reference aided method (pseudo de-novo).

Typical use cases involve multi-allelic samples where the sample-specific ploidy or copy number is unknown. _pbaa_ can effectively separate alleles with one to many variants, including SNVs and large indels contained within the target region. _pbaa_ has been optimized and tested for datasets with a moderate (<10) cluster count. Feedback for higher cluster density is welcome and may be addressed in future releases.
Typical use cases involve multi-allelic samples where the sample-specific ploidy or copy number is unknown. _pbaa_ can effectively separate alleles with one to many variants, including SNVs and large indels contained within the target region. _pbaa_ has been optimized and tested for datasets with a moderate (<10) cluster count. Feedback for higher cluster density is welcome and may be addressed in future releases.

## Workflow
![HiFi Amplicon Analysis Workflow](img/workflow.png)
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Guide/reference sequence choice affects read grouping/placement. It is important to choose guides that are sufficiently divergent. If too many similar alleles are used for the same locus the fraction of un-placed reads will increase because the number of informative kmers decrease within a locus. Too few guides can also cause cluster dropout; it's the goldilocks problem.

Guide sequences should be grouped into locus assignments. For example if multiple HLA-A alleles are used in the guide sequence, they should be grouped, so clustering will be performed at the locus level.
Guide sequences should be grouped into locus assignments. For example if multiple HLA-A alleles are used in the guide sequence, they should be grouped, so clustering will be performed at the locus level.

```
Allele_1|HLA-A (sequence name | group name)
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## Best practices

### Sample preparation and sequencing
### Sample preparation and sequencing

[Targeted Sequencing For Amplicons Document](https://www.pacb.com/wp-content/uploads/Application-Brief-Targeted-sequencing-Best-Practices.pdf)

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