diff --git a/PyHRM.py b/PyHRM.py index c66338c..43424ff 100644 --- a/PyHRM.py +++ b/PyHRM.py @@ -86,7 +86,17 @@ # # So you can identify your genotyping results by looking at: to which control they cluster. +# Ploting with plot.ly, so you can look at individual lines for better pattern recognition # In[ ]: +import plotly.plotly as py +import cufflinks as cf +import plotly.graph_objs as go +cf.set_config_file(offline=False, world_readable=True, theme='ggplot') + +dfpy = dfdif.set_index(df_melt.iloc[:,0]) + +# Plot and embed in ipython notebook! +dfpy.iplot(kind='scatter', filename='pyHRM') diff --git a/README.md b/README.md index 412e7ce..df5e542 100644 --- a/README.md +++ b/README.md @@ -7,6 +7,18 @@ You can view this ipython notebook demo here: https://github.com/liuyigh/PyHRM/blob/master/PyHRM.ipynb +## FAQ + +### Clustering not working. + +When you get noisy data, the k-means is not going to magically salvage it. Try these: + +* Do your PCR with touch down protocol, it greatly improves data quality, like magic! +* Make sure you get rid off empty wells, failed wells (look at your melting curve peaks), obvious outliers +* Make sure you choose the best temp range +/- 5 degree C around melting temp usually works the best. +* For subtle differences, your eyes can be better at pattern recognition than k-means. Use the provided code to plot it with `plot.ly`. You can look at individual lines on plot.ly to make your own judgement. +* Reduce heat block variation by running only 1 target gene in symatrically arranged wells. + ## How sensitive is pyHRM? I am able to reliably detect: @@ -18,6 +30,8 @@ I am able to reliably detect: ## qPCR protocol +Do your PCR with **touch down** protocol, it greatly improves data quality, like magic! + ## Basics: HRM - High Resolution Melt Analysis [Kapa BioSystems HRM Guide](http://www.kapabiosystems.com/document/introduction-high-resolution-melt-analysis-guide/)