Vignette¶

This vignette extends the vignette for the R-version of tximport. If you are unfamiliar with tximport or curious about the motivation behind it, please check it out.

If you are looking for a full-featured end-to-end workflow for Pythonic bulk RNA-sequencing analysis, check out our Snakemake workflow based on pytximport.

Creating your transcript to gene map¶

Here, we will show you how to generate a transcript-to-gene mapping based on the Ensembl reference or a gene transfer format file.

Build it from Ensembl¶

This example requires pybiomart which is installed together with pytximport. Providing a host is optional, for a list of available archives that correspond to Ensembl releases, please consult https://www.ensembl.org/info/website/archives/index.html. By default, the transcript ids will be mapped to the ensembl_gene_id field. If you prefer to use gene names, choose external_gene_name. Be aware that not all proposed transcripts have been assigned a name yet and thus will not be included if you use gene names. The first time you run this function, it may take a few seconds to download the data.

[1]:

from pytximport.utils import create_transcript_gene_map

transcript_gene_map_human = create_transcript_gene_map(
    species="human",
    target_field="external_gene_name",
)
transcript_gene_map_human.head(5)

[1]:

	transcript_id	gene_id
0	ENST00000387314	MT-TF
1	ENST00000389680	MT-RNR1
2	ENST00000387342	MT-TV
3	ENST00000387347	MT-RNR2
4	ENST00000386347	MT-TL1

[2]:

transcript_gene_map_mouse = create_transcript_gene_map(species="mouse")
transcript_gene_map_mouse.head(5)

[2]:

	transcript_id	gene_id
0	ENSMUST00000082387	ENSMUSG00000064336
1	ENSMUST00000082388	ENSMUSG00000064337
2	ENSMUST00000082389	ENSMUSG00000064338
3	ENSMUST00000082390	ENSMUSG00000064339
4	ENSMUST00000082391	ENSMUSG00000064340

Use a gene transfer format file¶

If you already have an annotation file in .gtf format (e.g. from the GENCODE or Ensembl references), you can use the create_transcript_gene_map_from_annotation function contained in pytximport.utils to generate a transcript to gene map. Using an annotation file is considered best practice, as it ensures that your annotation and alignment reference are the same if you download them from the same source, e.g., an Ensembl release.

Set target_field to gene_name to map transcript ids to gene names instead of gene ids. If the transcript does not have a corresponding gene name in the annotation file, it will be mapped to the gene id.

[3]:

from pytximport.utils import create_transcript_gene_map_from_annotation

transcript_gene_map_from_gtf = create_transcript_gene_map_from_annotation(
    "../../test/data/annotation.gtf",
    target_field="gene_name",
)
transcript_gene_map_from_gtf.head(5)

[3]:

	transcript_id	gene_id
0	ENST00000424215	ENSG00000228037
1	ENST00000511072	PRDM16
2	ENST00000607632	PRDM16
3	ENST00000378391	PRDM16
4	ENST00000514189	PRDM16

Map transcript names to gene names¶

You can also optionally map transcript names to either gene names or gene ids if your data requires it.

[4]:

transcript_name_gene_map_human = create_transcript_gene_map(
    "human",
    source_field="external_transcript_name",
    target_field="external_gene_name",
)
transcript_name_gene_map_human.head(5)

[4]:

	transcript_id	gene_id
0	MT-TF-201	MT-TF
1	MT-RNR1-201	MT-RNR1
2	MT-TV-201	MT-TV
3	MT-RNR2-201	MT-RNR2
4	MT-TL1-201	MT-TL1

Exporting transcript-level count estimates¶

You can also export the transformed transcript counts directly for transcript-level analysis.

[7]:

txi = tximport(
    ["../../test/data/salmon/quant.sf"],
    "salmon",
    counts_from_abundance="scaled_tpm",
    return_transcript_data=True,
)
txi

Reading quantification files: 1it [00:00, 323.11it/s]

[7]:

AnnData object with n_obs × n_vars = 1 × 14
    uns: 'counts_from_abundance'
    obsm: 'length', 'abundance'

Note that the example above works without a transcript to gene map. If you want to use the transcript names instead of the transcript ids, you can optionally use the replace_transcript_ids_with_names function together with a transcript id to transcript name map. We can use the create_transcript_gene_map function to create a map between transcript ids and transcript names, too.

[8]:

from pytximport.utils import replace_transcript_ids_with_names

[9]:

transcript_name_map_human = create_transcript_gene_map("human", target_field="external_transcript_name")
transcript_name_map_human.head(5)

[9]:

	transcript_id	transcript_name
0	ENST00000387314	MT-TF-201
1	ENST00000389680	MT-RNR1-201
2	ENST00000387342	MT-TV-201
3	ENST00000387347	MT-RNR2-201
4	ENST00000386347	MT-TL1-201

[10]:

txi = replace_transcript_ids_with_names(txi, transcript_name_map_human)
pd.DataFrame(txi.X.T, index=txi.var.index, columns=txi.obs.index).sort_values(
    by=txi.obs.index[0],
    ascending=False,
).head(5)

[10]:

	../../test/data/salmon/quant.sf
HOXC8-201	4486.940412
UGT3A2-201	1307.314695
HOXC9-201	886.909534
HOXC4-202	749.069369
HOXC12-201	544.817685

The same data summarized to genes:

[11]:

txi = tximport(
    ["../../test/data/salmon/quant.sf"],
    "salmon",
    transcript_gene_map_human,
    counts_from_abundance="scaled_tpm",
    output_type="xarray",
    return_transcript_data=False,
)
pd.DataFrame(txi["counts"], index=txi.coords["gene_id"], columns=txi.coords["file_path"]).sort_values(
    by=txi.coords["file_path"].data[0],
    ascending=False,
).head(5)

Reading quantification files: 1it [00:00, 114.91it/s]

[11]:

	../../test/data/salmon/quant.sf
HOXC8	4486.940412
UGT3A2	1506.597257
HOXC4	1152.964133
HOXC9	886.909534
HOXC12	544.817685

Note that the top transcript corresponds to the top expressed gene in this case.

Exporting AnnData files¶

pytximport integrates well with other packages from the scverse through its AnnData export option.

[12]:

txi_ad = tximport(
    ["../../test/data/salmon/quant.sf"],
    "salmon",
    transcript_gene_map_human,
    output_type="anndata",
    # the output can optionally be saved to a file by uncommenting the following lines
    # output_format="h5ad",
    # output_path="txi_ad.h5ad",
)
txi_ad

Reading quantification files: 1it [00:00, 332.99it/s]

[12]:

AnnData object with n_obs × n_vars = 1 × 10
    uns: 'counts_from_abundance'
    obsm: 'length', 'abundance'

Exporting SummarizedExperiment files¶

Experiment support for SummarizedExperiment files is available through the BiocPy ecosystem (unaffiliated). While not part of the core functionality of pytximport, this output type/format may be useful for interacting with other R software packages. The optional dependencies necessary to support SummarizedExperiment files can be installed via pip install pytximport[biocpy].

[13]:

txi_se = tximport(
    ["../../test/data/salmon/quant.sf"],
    "salmon",
    transcript_gene_map_human,
    output_type="summarizedexperiment",
    # the output can optionally be saved to disk by uncommenting the following lines
    # output_format="summarizedexperiment",
    # output_path="txi_se",
)
txi_se.assay_names, txi_se.get_row_names(), txi_se.get_column_names()

Reading quantification files: 1it [00:00, 372.03it/s]
WARNING:root:Support for the SummarizedExperiment output type is experimental.

[13]:

(['counts'],
 Names(['UGT3A2', 'HOXC6', 'HOXC9', 'HOXC11', 'HOXC4', 'HOXC10', 'HOXC13', 'HOXC5', 'HOXC8', 'HOXC12']),
 Names(['../../test/data/salmon/quant.sf']))

Use from the command line¶

You can run pytximport from the command line, too. Available options can be viewed via the pytximport --help command.

[14]:

!pytximport -i ../../test/data/salmon/quant.sf -t "salmon" -m ../../test/data/gencode.v46.metadata.HGNC.tsv -of "h5ad" -ow -o ../../test/data/salmon/quant.h5ad

2024-10-01 12:36:11,849: Starting the import.
Reading quantification files: 1it [00:00, 114.33it/s]
2024-10-01 12:36:12,137: Converting transcript-level expression to gene-level expression.
2024-10-01 12:36:12,239: Matching gene_ids.
2024-10-01 12:36:12,259: Creating gene abundance.
2024-10-01 12:36:12,409: Creating gene counts.
2024-10-01 12:36:12,409: Creating lengths.
2024-10-01 12:36:12,409: Replacing missing lengths.
2024-10-01 12:36:12,414: Creating gene expression dataset.
2024-10-01 12:36:12,415: Saving the gene-level expression to: ../../test/data/salmon/quant.h5ad.
2024-10-01 12:36:12,419: Finished the import in 0.57 seconds.

Inferential replicates¶

pytximport can handle bootstraping replicates provided by salmon and kallisto. When inferential_replicate_transformer is set, the provided function is used to recalculate the counts and abundances for each sample based on the bootstraps.

[15]:

result = tximport(
    [
        "../../test/data/fabry_disease/SRR16504309_wt/",
        "../../test/data/fabry_disease/SRR16504310_wt/",
        "../../test/data/fabry_disease/SRR16504311_ko/",
        "../../test/data/fabry_disease/SRR16504312_ko/",
    ],
    "salmon",
    transcript_gene_map_human,
    inferential_replicates=True,
    inferential_replicate_variance=True,  # whether to calculate the variance of the inferential replicates
    inferential_replicate_transformer=lambda x: np.median(x, axis=1),
    counts_from_abundance="length_scaled_tpm",
)
result

Reading quantification files: 4it [00:00,  9.55it/s]
WARNING:root:Not all transcripts are present in the mapping. 31380 out of 253181 missing. Removing the missing transcripts.

[15]:

AnnData object with n_obs × n_vars = 4 × 40768
    uns: 'counts_from_abundance', 'inferential_replicates'
    obsm: 'length', 'abundance', 'variance'

Downstream analysis with PyDESeq2¶

The output from pytximport can easily be used for downstream analysis with PyDESeq2. For more information on PyDESeq2, please consult its documentation.

[16]:

%pip install pydeseq2 numba==0.60.0 decoupler adjustText omnipath tqdm ipywidgets seaborn -q

Note: you may need to restart the kernel to use updated packages.

[17]:

from pydeseq2.dds import DeseqDataSet
from pydeseq2.default_inference import DefaultInference
from pydeseq2.ds import DeseqStats
import decoupler as dc

Load the .csv file generated by pytximport via the output_path argument or create it directly from the output of pytximport. In this case, we are working with the salmon quantification files from a public bulk RNA sequencing dataset: Podocyte injury in Fabry nephropathy

Round count estimates (required by PyDESeq2) and add the corresponding metadata.

[18]:

result.X = result.X.round().astype(int)
result.obs["condition"] = ["Control", "Control", "Disease", "Disease"]
result

[18]:

AnnData object with n_obs × n_vars = 4 × 40768
    obs: 'condition'
    uns: 'counts_from_abundance', 'inferential_replicates'
    obsm: 'length', 'abundance', 'variance'

Filter genes with low counts out.

[19]:

result = result[:, result.X.max(axis=0) > 10].copy()
result

[19]:

AnnData object with n_obs × n_vars = 4 × 14714
    obs: 'condition'
    uns: 'counts_from_abundance', 'inferential_replicates'
    obsm: 'length', 'abundance', 'variance'

Now perform your PyDESeq2 analysis.

[20]:

dds = DeseqDataSet(
    adata=result,
    design_factors="condition",
    refit_cooks=True,
    inference=DefaultInference(n_cpus=8),
    quiet=True,
)

[21]:

dds.deseq2()

/Users/au734063/Documents/code/pytximport-publish/pytximport/.venv/lib/python3.12/site-packages/pydeseq2/dds.py:490: UserWarning: As the residual degrees of freedom is less than 3, the distribution of log dispersions is especially asymmetric and likely to be poorly estimated by the MAD.
  self.fit_dispersion_prior()

[22]:

stat_result = DeseqStats(dds, contrast=("condition", "Disease", "Control"), quiet=True)

[23]:

%%capture
stat_result.summary()
stat_result.lfc_shrink()

[24]:

dc.plot_volcano_df(
    stat_result.results_df,
    x="log2FoldChange",
    y="padj",
    sign_thr=0.01,
    top=20,
    figsize=(10, 5),
)

[25]:

collectri = dc.get_collectri(organism="human", split_complexes=False)
mat = stat_result.results_df[["stat"]].T.rename(index={"stat": "disease.vs.control"})
tf_acts, tf_pvals = dc.run_ulm(mat=mat, net=collectri, verbose=True)
dc.plot_barplot(acts=tf_acts, contrast="disease.vs.control", top=10, vertical=False, figsize=(5, 3))

Running ulm on mat with 1 samples and 14714 targets for 660 sources.

We can also evaluate known pathways.

[26]:

progeny = dc.get_progeny(top=500)
pathway_acts, pathway_pvals = dc.run_mlm(mat=mat, net=progeny, min_n=5)

[27]:

dc.plot_barplot(
    acts=pathway_acts,
    contrast="disease.vs.control",
    top=40,
    vertical=False,
    figsize=(5, 3),
)

Please refer to the PyDESeq2 and the decoupler documentation for additional analyses.