sanjaynagi / anoprimer

Primer Design in Anopheles gambiae and funestus considering genetic variation

Primer design in Anopheles gambiae s.l and funestus taking into account SNP variation in primer binding sites, using primer3-py and malariagen_data. Supports genomic DNA primers, cDNA primers and hybridisation probes:

Documentation: https://sanjaynagi.github.io/AnoPrimer/

anoprimer's Issues

Cannot install on Python 3.10

Currently AgamPrimer cannot be installed on Python 3.10 or above because of the pinning of the Python dependency.

This is particularly a problem for colab users because colab has recently been upgraded to Python 3.10.

add support for CNVs

Need to figure out the possible CNV orientations. this would possibly require manually providing the cnv sequence and breakpoints

make a faq on the documentation

how do I design a primer to target different loci of the gene?
For gDNA primers, you specify the exact genomic location in base pairs - ie. 2L:2,000,000
Is it possible to specify the exon or intergenic region when designing the primer?
for gDNA primers, yes, for cDNA primers, they will target exon-exon junctions
How can I check the primer's dimerisation probality if I have designed several primers?
Primer 3 will do this internally within AgamPrimer. If you want to see dimerisation results, you have to use the long AgamPrimer notebook, and look at the 'primer_dict' otherwise i recommend tools on IDT website
how can I specify the primers binding regions for example exon 1 to exon 4 with a single pair primer?
you cant but you can play around with the amplicon preferred size and target until it does so
In one of the exercises, SNPs were found only on the probe sequence, could this affect the efficiency of the qpcr?
It depends where in the probe and what kind of assay you are doing. you havent mentioned?

Forbidden control characters in expression in Workshop 6's module 4

I was rerunning the notebook from Workshop 6 Module 4 and it raised the following error:

I gather there is a parsing issue with some of the parameters but I haven't really looked at the code yet.

make exon-exon junctions for cDNA optional

add funestus support

Through malariagen_data.Af1.

• At which point we should rename to AnoPrimer
• does gget BLAT database have funestus?

optimise AgamPrimer with snakeviz and numba

Where/if appropriate

Currently plot_primer_ag3_frequencies() is probably the only function worth optimising.

A scoring function for oligo binding

So we can rank primer pairs on the SNP-variation score

Declare dependencies in package metadata

AgamPrimer currently depends on several packages:

import pandas as pd 
import allel 
import numpy as np
import seaborn as sns
import matplotlib.pyplot as plt
from matplotlib import patches
import malariagen_data
from plotly.subplots import make_subplots
import plotly.graph_objects as go
import gget
import primer3

It would be good to express these dependencies in the package metadata so that they get installed automatically when AgamPrimer is installed via pip.

Not all of these would need to be stated directly, as several will get brought in as transitive dependencies via malariagen_data. However, not all will, e.g., at least seaborn, gget and primer3 would need to be stated explicitly.

There are several different ways to state dependencies.

If using poetry, these can be declared in the pyproject.toml file, see an example.

The old-school way to do it is by adding an install_requires into setup.py.

report exact alternate alleles

Report exact alternate bases for A,C, G, T, rather than summing the frequency of all alts.

And suggest degenerate bases for those primers

add docstrings to all public functions, make jupyter book

report sample size of Ag1000g pops

In title of figure

rename qPCR to cDNA

'qPCR primers' is misleading, because qPCR assays can be done on genomic DNA, as well as cDNA.

We really mean cDNA primers here, as one primer is targeting an exon-exon junction, it will only amplify cDNA. This will also align with gDNA primers.

Change target argument to region string

so target either is '2L:28545767' or 'AGAP006227-RA'

Remove the need to provide contig, which is unnecessary anyway for cDNA primers in which the transcript is provided.

add support to check pre-existing primers

possible with gget BLAT to find genomic location and then malariagen_data

add sample query option to Ag1000g pop selection

So one can filter for species etc

allow the design of cDNA primers + probe

For use in for example, taqman or lna probe for gene expression, where you have a single probe and primers.

add blast support with gget

add tests and CI with github actions

Add support for primer3-py 2.0.0

in long notebook move ag1000g cohort choice in colab form at beginning

add genomic primer span to primer df

allow user to configure paths to save outputs to

support for probes

Support to design internal oligos (for example, the base sequence for designing LNA probes)