# Exploring copy number variation (CNV)¶

In this notebook we will explore copy number variation (CNV) within the Ag1000G phase 3 samples, focusing on some specific genome regions of interest. We will use the copy number state data for each individual sample, inferred from fitting an HMM to windowed coverage data, to visualise evidence for copy number variation. We will also estimate the frequency of copy number variation in different genes and populations.

As usual this notebook is executable, click the launch icon above () to run it for yourself. Note in particular that, although this notebook investigates the Cyp9k1 and Cyp6p/aa genome regions as examples, if you run this notebook yourself you can change this to investigate any genome region you are interested in.

## Setup¶

Install the packages we’ll need. If you’re working on MyBinder, you can skip this step, packages are already installed.

!pip install -qU malariagen_data bokeh


Import packages.

import malariagen_data
from bisect import bisect_left, bisect_right
import numpy as np
import bokeh.io as bkio
import pandas as pd
import matplotlib.pyplot as plt
%matplotlib inline


In this notebook we’ll be making some interactive plots using the bokeh package. Set up plotting with bokeh in the notebook.

bkio.output_notebook()


Finally, setup access to Ag3 data in Google Cloud.

ag3 = malariagen_data.Ag3()
ag3

MalariaGEN Ag3 data resource API
Storage URL gs://vo_agam_release/
Data releases available 3.0
Cohorts analysis 20211101
Species analysis aim_20200422
Site filters analysis dt_20200416
Software version 3.0.0

## Introduction to copy number variation¶

Mosquitoes are diploid organisms, like us, meaning that each individual mosquito inherits two complete genome copies, one from each parent. Thus, for any given gene within the mosquito genome sequence, an individual will normally carry two copies.

Of course, genomes can vary. One common type of genome variation is single nucleotide polymorphism (SNP), which we’ve previously explored in the analysis notebooks on SNPs and SNP effects. Another way that genomes can vary is where a gene, or a genome region containing multiple genes, becomes copied two or more times within the same chromosome. Genes or genome regions can also sometimes be deleted. This is known as copy number variation (CNV).

Previously, the Ag1000G Consortium analysed copy number variation within the Ag1000G phase 2 data resource (Lucas et al. 2019). A key finding was that, although CNVs can occur throughout the mosquito genome, there are several genome hotspots where CNVs occur much more frequently. Several of these hotspots occur in genome regions containing genes involved in metabolic resistance to insecticides, such as cytochrome P450 and glutathione S-transferase genes. It has been known for several decades that copy number variation within these gene families likely plays an important role in insecticide resistance. More copies of a gene means more of the corresponding protein will be expressed, and increasing expression of genes that can metabolise insecticides leads to increased ability to tolerate the insecticide (Adolfi et al. 2019).

For the Ag1000G phase 3 data resource, we have estimated the copy number state for each individual mosquito throughout the entire genome sequence, compared to the AgamP4 reference sequence. For each mosquito we’ve counted the number of aligned sequence reads within fixed-size 300 bp windows, then normalised this coverage data and fitted a hidden Markov model. Further details are given in the section on CNV calling in the methods page.

The results of these CNV HMMs provide a rich dataset for exploring copy number variation in any given genome region of interest.

## Accessing CNV HMM data¶

The CNV HMM results for a given chromosome and given sample sets can be accessed using the cnv_hmm method. Let’s access the data for chromosome arm 2R, for two of the Ag3 sample sets from Burkina Faso.

ds_hmm = ag3.cnv_hmm(region="2R", sample_sets=["AG1000G-BF-A", "AG1000G-BF-B"])
ds_hmm

<xarray.Dataset>
Dimensions:                   (samples: 283, variants: 205151)
Coordinates:
Dimensions without coordinates: samples, variants
Data variables:
call_CN                   (variants, samples) int8 dask.array<chunksize=(65536, 64), meta=np.ndarray>
call_RawCov               (variants, samples) int32 dask.array<chunksize=(65536, 64), meta=np.ndarray>
call_NormCov              (variants, samples) float32 dask.array<chunksize=(65536, 64), meta=np.ndarray>
Attributes:
contigs:  ('2R', '2L', '3R', '3L', 'X')

The ds_hmm variable is an xarray dataset. This is simply a container for several data arrays. In particular, we’ll be most interested in the variant_position and variant_end arrays, which provide the genomic coordinates for the window start and stop positions; and the call_CN array, which provides the inferred copy number state across samples and windows. Let’s look at each of these in turn.

# window start coordinates (1-based)
ds_hmm['variant_position'].values

array([       1,      301,      601, ..., 61544401, 61544701, 61545001],
dtype=int32)

# window end coordinates (1-based, inclusive)
ds_hmm['variant_end'].values

array([     300,      600,      900, ..., 61544700, 61545000, 61545105],
dtype=int32)

# copy number state predictions from the HMMs
ds_hmm['call_CN']

<xarray.DataArray 'call_CN' (variants: 205151, samples: 283)>
dask.array<concatenate, shape=(205151, 283), dtype=int8, chunksize=(65536, 64), chunktype=numpy.ndarray>
Coordinates:
Dimensions without coordinates: variants, samples

Notice that the call_CN array has 205,151 rows and 283 columns. That’s because there are 205,151 windows on chromosome arm 2R, and we’ve requested data for 283 individual mosquitoes. For example, here are the copy number state predictions for the first 5 genome windows, for the first sample:

cn = ds_hmm['call_CN'].values
cn[:5, 0]

array([7, 4, 4, 8, 8], dtype=int8)


The numbers 7, 4, 4, 8 and 8 are copy number states. Above I said that the normal copy number state is 2, so what’s going on here? Well, these values are from the beginning of the chromosome arm, and the ends of the chromosome can exhibit a lot of copy number variation. Let’s gain some more intuition by making a histogram of the call_CN values across all windows and individuals.

fig, ax = plt.subplots()
ax.hist(cn.flatten(), bins=np.arange(-1, 13, 1))
ax.set_xlabel('Copy number state')
ax.set_ylabel('Frequency')
ax.set_title('Copy number histogram')
ax.set_xticks(np.arange(-1, 13, 1) + .5)
ax.set_xticklabels(np.arange(-1, 13, 1));


Above we can see that by far the most common copy number state is 2, as expected. A copy number state less than 2 implies a deletion relative to the reference genome. A copy number state greater than 2 implies an amplification relative to the reference genome. The special value -1 means that the copy number state could not be inferred because of unreliable sequence read alignments.

OK, now we have some sense of what the data look like, let’s explore some specific genome regions of interest.

## Visualising amplifications and deletions¶

There are a number of ways of plotting the results of the CNV HMMs in order to visually search for the presence or absence of CNVs in a genome region of interest, and explore their locations and frequencies in different populations.

### Cyp9k1 region¶

In our previous analysis of Ag1000G phase 2, we found that the region surrounding the Cyp9k1 gene on the X chromosome was a CNV hotspot. Let’s explore that in the Ag3 data resource.

ag3.plot_cnv_hmm_heatmap(
region="X:15,233,000-15,260,000",
sample_sets=["AG1000G-BF-A", "AG1000G-BF-B"],
sample_query="taxon == 'gambiae' and sex_call == 'F'"
);


In the above plots, we can see that many of the An. gambiae individuals have a copy number amplification spanning the Cyp9k1 gene.

Let’s compare with the An. coluzzii.

ag3.plot_cnv_hmm_heatmap(
region="X:15,233,000-15,260,000",
sample_sets=["AG1000G-BF-A", "AG1000G-BF-B"],
sample_query="taxon == 'coluzzii' and sex_call == 'F'"
);


Apparently, amplifications spanning Cyp9k1 in the An. coluzzii in these sample sets are much less common.

### Cyp6p/aa region¶

For comparison, let’s also visualise CNVs in the Cyp6p/aa region on chromosome arm 2R, another known CNV hotspot.

ag3.plot_cnv_hmm_heatmap(
region="2R:28,450,000-28,580,000",
sample_sets=["AG1000G-BF-A", "AG1000G-BF-B"],
sample_query="taxon == 'gambiae'"
);


Again, let’s compare with the An. coluzzii from the same sample sets.

ag3.plot_cnv_hmm_heatmap(
region="2R:28,450,000-28,580,000",
sample_sets=["AG1000G-BF-A", "AG1000G-BF-B"],
sample_query="taxon == 'coluzzii'"
);


In this genome region, amplifications appear to be much more common among the An. coluzzii in these sample sets than the An. gambiae. This is an indication that, within Burkina Faso, different cytochrome P450 genes may be playing a role in pyrethroid insecticide resistance in different species.

## Summarising CNV frequencies by gene¶

In addition to visualising the data, it can also be useful to summarise the HMM results by gene. In this case, we might use the modal copy number state within the windows spanning a gene to estimate the gene copy number within an individual mosquito. The gene_cnv_frequencies method provides an implementation of this.

Let’s illustrate this by computing CNV frequencies for the genes in the Cyp6p/aa cluster, again using two Burkina Faso sample sets. These two sample sets provide data from two different species and time points, which can be intersting to compare.

Compute CNV frequencies in genes in the Cyp6aa/p cluster.

df_cnv_cyp6aap = ag3.gene_cnv_frequencies(
region="2R:28,450,000-28,510,000",
sample_sets=["AG1000G-BF-A", "AG1000G-BF-B"]
)
df_cnv_cyp6aap

gene_strand gene_description contig start end frq_BF-09_colu_2012 frq_BF-09_colu_2014 frq_BF-09_gamb_2012 frq_BF-09_gamb_2014 max_af windows label
gene_id gene_name cnv_type
AGAP002862 CYP6AA1 amp - cytochrome P450 [Source:VB Community Annotation] 2R 28480576 28482637 0.9125 0.811321 0.041237 0.065217 0.912500 8 AGAP002862 (CYP6AA1) amp
AGAP013128 CYP6AA2 amp - cytochrome P450 [Source:VB Community Annotation] 2R 28483301 28484921 0.8250 0.792453 0.041237 0.065217 0.825000 6 AGAP013128 (CYP6AA2) amp
AGAP002863 COEAE6O amp - carboxylesterase alpha esterase [Source:VB Com... 2R 28485262 28487080 0.6000 0.509434 0.010309 0.021739 0.600000 7 AGAP002863 (COEAE6O) amp
AGAP002864 CYP6P15P amp - cytochrome P450 [Source:VB Community Annotation] 2R 28487640 28489092 0.5250 0.528302 0.010309 0.021739 0.528302 6 AGAP002864 (CYP6P15P) amp
AGAP002865 CYP6P3 amp - cytochrome P450 [Source:VB Community Annotation] 2R 28491415 28493141 0.0375 0.075472 0.010309 0.021739 0.075472 7 AGAP002865 (CYP6P3) amp
del - cytochrome P450 [Source:VB Community Annotation] 2R 28491415 28493141 0.0000 0.000000 0.113402 0.000000 0.113402 7 AGAP002865 (CYP6P3) del
AGAP002866 CYP6P5 amp - cytochrome P450 [Source:VB Community Annotation] 2R 28494017 28495645 0.0375 0.056604 0.020619 0.021739 0.056604 6 AGAP002866 (CYP6P5) amp
AGAP002867 CYP6P4 amp - cytochrome P450 [Source:VB Community Annotation] 2R 28497087 28498674 0.0375 0.075472 0.010309 0.021739 0.075472 6 AGAP002867 (CYP6P4) amp
AGAP002868 CYP6P1 amp - cytochrome P450 [Source:VB Community Annotation] 2R 28499251 28500900 0.0375 0.056604 0.010309 0.021739 0.056604 6 AGAP002868 (CYP6P1) amp
AGAP002869 CYP6P2 amp - cytochrome P450 [Source:VB Community Annotation] 2R 28501033 28502910 0.0375 0.056604 0.010309 0.021739 0.056604 7 AGAP002869 (CYP6P2) amp
AGAP002870 CYP6AD1 amp - cytochrome P450 [Source:VB Community Annotation] 2R 28504248 28505816 0.0375 0.056604 0.010309 0.021739 0.056604 6 AGAP002870 (CYP6AD1) amp

Finally, let’s make this table a little more readable by removing some columns and highlighting frequencies.

ag3.plot_frequencies_heatmap(df_cnv_cyp6aap)


Here we can see that the most commonly amplified gene is Cyp6aa1, which is amplified in 90% of An. coluzzii individuals in 2012 and 81% of An. coluzzii individuals in 2014.

Hopefully this notebook has been a useful introduction to exploring CNVs in the Ag3 data resource. Don’t forget you can use this notebook to analyse any genome region of interest. If you have any comments, questions or suggestions, please feel free to get in touch.