Follow the Data

A data driven blog

Archive for the tag “tutorial”

Tutorial: Exploring TCGA breast cancer proteomics data

Data used in this publication were generated by the Clinical Proteomic Tumor Analysis Consortium (NCI/NIH).

The Cancer Genome Atlas (TCGA) has become a focal point for a lot of genomics and bioinformatics research. DNA and RNA level data on different tumor types are now used in countless papers to test computational methods and to learn more about hallmarks of different types of cancer.

Perhaps, though, there aren’t as many people who are using the quantitative proteomics data hosted by Clinical Proteomic Tumor Analysis Consortium (CPTAC). There are mass spectrometry based expression measurements for many different types of tumor available at their Data Portal.

As I have been comparing some (currently in-house, to be published eventually) cancer proteomics data sets against TCGA proteomics data, I thought I would share some code, tricks and tips for those readers who want to start analyzing TCGA data (whether proteomics, transcriptomics or other kinds) but don’t quite know where to start.

To this end, I have put a tutorial Jupyter notebook at Github: TCGA protein tutorial

The tutorial is written in R, mainly because I like the TCGA2STAT and Boruta packages (but I just learned there is a Boruta implementation in Python as well.) If you think it would be useful to have a similar tutorial in Python, I will consider writing one.

The tutorial consists, roughly, of these steps:

  • Getting a usable set of breast cancer proteomics data
    This consists of downloading the data, selecting the subset that we want to focus on, removing features with undefined values, etc..
  • Doing feature selection to find proteins predictive of breast cancer subtype.
    Here, the Boruta feature selection package is used to identify a compact set of proteins that can predict the so-called PAM50 subtype of each tumor sample. (The PAM50 subtype is based on mRNA expression levels.)
  • Comparing RNA-seq data and proteomics data on the same samples.
    Here, we use the TCGA2STAT package to obtain TCGA RNA-seq data and find the set of common gene names and common samples between our protein and mRNA-seq data in order to look at protein-mRNA correlations.

Please visit the notebook if you are interested!

Some of the take-aways from the tutorial may be:

  • A bit of messing about with metadata, sample names etc. is usually necessary to get the data in the proper format, especially if you are combining different kinds of data (such as RNA-seq and proteomics here). I guess you’ve heard them say that 80% of data science is data preparation!…
  • There are now quantitative proteomics data available for many types of TCGA tumor samples.
  • TCGA2STAT is a nice package for importing certain kinds of TCGA data into an R session.
  • Boruta is an interesting alternative for feature selection in a classification context.

This post was prepared with permission from CPTAC.

P.S. I may add some more material on a couple of ways to do multivariate data integration on TCGA data sets later, or make that a separate blog post. Tell me if you are interested.

Notes on genomics APIs #3: SolveBio

This is the third in a short series of posts with notes on different genomics APIs. The first post, which was about the One Codex API, can be found here, and the second one, about Google Genomics, can be found here.

SolveBio “delivers the critical reference data used by hospitals and companies to run genomic applications”, according to their web page. They focus on clinical genomics and on helping developers who need to access various data sources in a programmatic way. Their curated data library provides access to (as of February 2015) “over 300 datasets for genomics, proteomics, literature annotation, variant-disease relationships, and more.) Some examples of those datasets are the ClinVar disease gene database from NIH, the Somatic Mutations dataset from The Cancer Genome Atlas, and the COSMIC catalogue of somatic mutations in cancer.

SolveBio offers a RESTful API with Python and Ruby clients already available and an R client under development. The Getting Started Guide really tells you most of what you need to know to use it, but let’s try it out here on this blog anyway!

You should, of course, start by signing up for a free account. After that, it’s time to get the client. I will use the Python one in this post. It can be installed by giving this command:

curl -skL | bash

You can also install it with pip.

Now you will need to login. This will prompt you for your email and password that you registered when signing up.

solvebio login

At this point you can view a useful tutorial by giving solvebio tutorial. The tutorial explains the concept of depositories, which are versioned containers for data sets. For instance (as explained in the docs), there is a ClinVar depository which (as of version 3.1.0) has three datasets: ClinVar, Variants, and Submissions. Each dataset within a depository is designed for a specific use-case. For example, the Variants dataset contains data on genomic variants, and supports multiple genome builds.

Now start the interactive SolveBio shell. This shell (in case you followed the instructions above) is based on iPython.


The command Depository.all() will show the available depositories. Currently, the list looks like this (you’ll want to click the image to blow it up a bit):
Screen Shot 2015-02-04 at 15.29.50

In a similar way, you can view all the data sets with Dataset.all(). Type Dataset.all(latest=True) to view only the latest additions.

To work with a data set, you need to ‘retrieve’ it with a command like:

ds = Dataset.retrieve('ClinVar/3.1.0-2015-01-13/Variants')

It is perfectly possible to leave out the version of the data set: ds = Dataset.retrieve('ClinVar/Variants') but that is bad practice from a reproducibility viewpoint and is not recommended, especially in production code.

Now we can check which fields are available in the ds object representing the data set we selected.


There are fields for things like alternate alleles for the variant in question, sources of clinical information on the variant, the name of any gene(s) overlapping the variant, and the genomic coordinates for the variant.

You can create a Python iterator for looping through all the records (variants) using ds.query(). To view the first variant, type ds.query()[0]. This will give you an idea of how each record (variant) is described in this particular data set. In practice, you will almost always want to filter your query according to some specified criteria. So for example, to look for known pathogenic variants in the titin (TTN) gene, you could filter as follows:

ttn_vars = ds.query().filter(clinical_significance='Pathogenic', gene_symbol_hgnc='TTN')

This will give you an iterator with a bunch of records (currently 18) that you can examine in more detail.

If you want to search for variants in some specified genomic region that you have identified as interesting, you can do that too, but it is only possible for some data sets. In this case it turns out that we can do it with this version of the ClinVar variant data set, because it is considered a “genomic” data set, which we can see because the command ds.is_genomicreturns True. (Some of the older versions return False here.)

ds.query(genome_build='GRCh37').range('chr3', 22500000, 23000000)

Note that you can specify a genome build in the query, which is very convenient.

Moving on to a different depository and data set, we can search for diabetes-related variants as defined via genome wide association studies with something like the following:

ds = Dataset.retrieve('GWAS/1.0.0-2015-01-13/GWAS')
ds.fields() # Check out which fields are available
ds.query().filter(phenotype='diabetes') # Also works with "Diabetes"
ds.query().filter(journal='science',phenotype='diabetes') # Only look for diabetes GWAS published in Science

Also, giving a command likeDataset.retrieve('GWAS/1.0.0-2015-01-13/GWAS').help() will open up a web page describing the dataset in your browser.

Notes on genomics APIs #2: Google Genomics API

This is the second in a series of about three posts with notes on different genomics APIs. The first post, which was about the One Codex API, can be found here.

As you may have heard, Google has started building an ambitious infrastructure for storing and querying genomic data, so I was eager to start exploring it. However, as there were a number of tools available, I initially had some trouble wrapping my head around what I was supposed to do. I hope these notes, where I mainly use the API for R, can provide some help.

Some useful bookmarks:

Google Developers Console – for creating and managing Google Genomics and BigQuery projects.

Google Genomics GitHub repo

Google Cloud Platform Google Genomics page (not sure what to call this page really)

Getting started

You should start by going to the Developer Console and creating a project. You will need to give it a name, and in addition it will be given a unique ID which you can use later in API calls. When the project has been created, click “Enable an API” on the Dashboard page, and click the button where it says “OFF” next to Genomics API (you may need to scroll down to find it).

Now you need to create a client_secret.json file that you will use for some API calls. Click the Credentials link in the left side panel and then click “Create new client ID”. Select “Installed application” and fill in the “Consent screen” form. All you really need to do is select an email address and type a “product name”, like “BlogTutorial” like I did for this particular example. Select “Installed application” again if you are prompted to select an application type. Now it should display some information under the heading “Client ID for native application”. Click the “Download JSON” button and rename the file to client_secret.json. (I got these instructions from here.)

Using the Java API client for exploring the data sets

One of the first questions I had was how to find out which datasets are actually available for querying. Although it is perfectly possible to click around in the Developer Console, I think the most straightforward way currently is to use the Java API client. I installed it from the Google Genomics GitHub repo by cloning:
git clone
The GitHub repo page contains installation instructions, but I will repeat them here. You need to compile it using Maven:

cd api-client-java
mvn package

If everything goes well, you should now be able to use the Java API client to look for datasets. It is convenient (but not necessary) to put the client_secret.json file into the same directory as the Java API client. Let’s check which data sets are available (this will only work for projects where billing has been enabled; you can sign up for a free trial in which case you will not be surprise-billed):

java -jar genomics-tools-client-java-v1beta2.jar listdatasets --project_number 761052378059 --client_secrets_filename client_secret.json

(If your client_secret.json file is in another directory, you need to give the full path to the file, of course.) The project number is shown on your project page in the Developer Console. Now, the client will open a browser window where you need to authenticate. You will only need to do this the first time. Finally, the results are displayed. They currently look like this:

Platinum Genomes (ID: 3049512673186936334)
1000 Genomes - Phase 3 (ID: 4252737135923902652)
1000 Genomes (ID: 10473108253681171589)

So there are three data sets. Now let’s check which reference genomes are available:

java -jar genomics-tools-client-java-v1beta2.jar searchreferencesets --client_secrets_filename ../client_secret.json --fields 'referenceSets(id,assemblyId)'

The output is currently:


To find out the names of the chromosomes/contigs in one of the reference genomes: (by default this will only return the ten first hits, so I specify –count 50)

java -jar genomics-tools-client-java-v1beta2.jar searchreferences --client_secrets_filename client_secret.json  --fields 'references(id,name)' --reference_set_id EMWV_ZfLxrDY-wE --count 50

Now we can try to extract a snippet of sequence from one of the chromosomes. Chromosome 9 in hg19 had the ID EIeX4KDCl634Jw, so the query becomes, if we want to extract some sequence from 13 Mbases into the chromosome:

java -jar genomics-tools-client-java-v1beta2.jar getreferencebases  --client_secrets_filename client_secret.json --reference_id ENywqdu-wbqQBA --start 13000000 --end 13000070


Another thing you might want to do is to check which “read groups” that are available in one of the data sets. For instance, for the Platinum Genomes data set we get:

java -jar genomics-tools-client-java-v1beta2.jar searchreadgroupsets --dataset_id 3049512673186936334  --client_secrets_filename client_secret.json

which outputs a bunch of JSON records that show the corresponding sample name, BAM file, internal IDs, software and version used for alignment to the reference genome, etc.

Using BigQuery to search Google Genomics data sets

Now let’s see how we can call the API from R. The three data sets mentioned above can be queried using Google’s BigQuery interface, which allows SQL-like queries to be run on very large data sets. Start R and install and load some packages:

install.packages("devtools") # unless you already have it!

Now we can access BigQuery through R. Try one of the non-genomics data sets just to get warmed up.

project <- '(YOUR_PROJECT_ID)' # the ID of the project from the Developer Console
sql <- 'SELECT title,contributor_username,comment FROM[publicdata:samples.wikipedia] WHERE title contains "beer" LIMIT 100;'
data <- query_exec(sql, project)

Now the data object should contain a list of Wikipedia articles about beer. If that worked, move on to some genomic queries. In this case, I decided I wanted to look at the SNP for the photic sneeze reflex (the reflex that makes people such as myself sneeze when they go out on a sunny day) that 23andme discovered via their user base. That genetic variant has the ID and is located on chromosome 2, base 146125523 in the hg19 reference genome. It seems that 23andme uses a 1-based coordinate system (the first nucleotide has the index 1) while Google Genomics uses a 0-based system, so we should look for base position 146125522 instead. We query the Platinum Genomes variant table: (you can find the available tables at the BigQuery Browser Tool Page)

sql <- 'SELECT reference_bases,alternate_bases FROM[genomics-public-data:platinum_genomes.variants] WHERE reference_name="chr2" AND start=146125522 GROUP BY reference_bases,alternate_bases;'
query_exec(sql, project)

This shows the following output:

reference_bases alternate_bases
1 C T

This seems to match the description provided by 23andme; the reference allele is C and the most common alternate allele is T. People with CC have slightly higher odds of sneezing in the sun, TT people have slightly lower odds, and people with CT have average odds.

If we query for the variant frequencies (VF) in the 13 Platinum genomes, we get the following results (the fraction represents, as I interpret it, the fraction of sequencing reads that has the “alternate allele”, in this case T):

sql <- 'SELECT call.call_set_name,call.VF FROM[genomics-public-data:platinum_genomes.variants] WHERE reference_name="chr2" AND start=146125522;'
query_exec(sql, project)

The output is as follows:

call_call_set_name call_VF
1 NA12882 0.500
2 NA12877 0.485
3 NA12889 0.356
4 NA12885 1.000
5 NA12883 0.582
6 NA12879 0.434
7 NA12891 1.000
8 NA12888 0.475
9 NA12886 0.434
10 NA12884 0.459
11 NA12893 0.588
12 NA12878 0.444
13 NA12892 0.533

So most people here seem to have a mix of C and T, with two individuals (NA12891 and NA12885) having all T:s, in other words they appear to be homozygous for the T allele, if I am interpreting this correctly.

Using the R API client

Now let’s try to use the R API client. In R, install the client from GitHub, and also ggbio and ggplot2 if you don’t have them already:


First we need to authenticate for this R session:

authenticate(file="/path/to/client_secret.json") # substitute the actual path to your client_secret.json file

The Google Genomics GitHub repo page has some examples on how to use the R API. Let’s follow the Plotting Alignments example.

reads <- getReads(readGroupSetId="CMvnhpKTFhDyy__v0qfPpkw",

This will fetch reads corresponding to the given genomic interval (which turns out to overlap a gene called KL) in the read group set called CMvnhpKTFhDyy__v0qfPpkw. By applying one of the Java API calls shown above and grepping for this string, I found out that this corresponds to a BAM file for a Platinum Genomes sample called NA12893.

We need to turn thereadslist into a GAlignment object:

alignments <- readsToGAlignments(reads)

Now we can plot the read coverage over the region using some ggbio functions.

alignmentPlot <- autoplot(alignments, aes(color=strand,fill=strand))
coveragePlot <- ggplot(as(alignments, 'GRanges')) + stat_coverage(color="gray40", fill="skyblue")
tracks(alignmentPlot, coveragePlot, xlab="Reads overlapping for NA12893")

As in the tutorial, why not also visualize the part of the chromosome where we are looking.

ideogramPlot <- plotIdeogram(genome="hg19", subchr="chr13")
ideogramPlot + xlim(as(alignments, 'GRanges'))


Now you could proceed with one of the other examples, for instance the variant annotation comparison example, which I think is a little bit too elaborate to reproduce here.

Notes on genomics APIs #1: One Codex

This is the first in a series of about three posts with notes on different genomics APIs.

One Codex calls itself “a genomic search engine, enabling new and valuable applications in clinical diagnostics, food safety, and biosecurity”. They have built a data platform where you can rapidly (much more quickly than with e.g. BLAST) match your sequences against an indexed reference database containing a large collection of bacterial, viral and fungal genomes. They have a good web interface for doing the search but have also introduced an API. I like to use command-line APIs in order to wrap things into workflows, so I decided to try it. Here are some notes on how you might use it.

This service could be useful when you want to identify contamination or perhaps the presence of some infectious agent in a tissue sample, but the most obvious use case is perhaps for metagenomics (when you have sequenced a mixed population of organisms). Let’s go to to the EBI Metagenomics site, which keeps a directory of public metagenomics data sets. Browsing through the list of projects, we see an interesting looking one: the Artisanal Cheese Metagenome. Let’s download one of the sequence files for that. Click the sample name (“Artisanal cheeses”), then click the Download tab. Now click “Submitted nucleotide reads (ENA website)”. There are two gzipped FASTQ files here – I arbitrarily choose to download the first one [direct link]. This download is 353 Mb and took about 10 minutes on my connection. (If you want a lighter download, you could try the 100 day old infant gut metagenome which is only about 1 Mb in size.)

The artisanal cheese metagenome file contains about 2 million sequences. If you wanted to do this analysis properly, you would probably want to run some de novo assembly tool which is good at metagenomics assembly such as IDBA-UD, Megahit, etc on it, but since my aim here is not to do a proper analysis but just show how to use the One Codex API, I will just query One Codex with the raw sequences.

I am going to use the full data set of 2M sequences. However, if you want to select a subset of let’s say 10,000 sequences in order to get results a bit faster, you could do like this:

gzcat Sample2a.fastq.gz | tail +4000000 | head -40000 > cheese_subset.fastq

(Some explanation is in order. In a FASTQ file, each sequence entry consists of four lines. Thus, we want to pick 40,000 lines in order to get 10,000 sequences. The tail +4000000 part of the command makes the selection start 1 million sequences into the file, that is, at 4 million lines. I usually avoid taking the very first sequences when choosing subsets of FASTQ files, because there are often poor sequences there due to edge effects in the sequencer flow cells. So now you would have selected 10,000 sequences from approximately the middle of the file.)

Now let’s try to use One Codex to see what the artisanal cheese metagenome contains. First, you need to register for a One Codex account, and then you need to apply for an API key (select Request a Key from the left hand sidebar).

You can use the One Codex API via curl, but there is also a convenient Python-based command-line client, which, however, only seems to work with Python 2 so far (a Python 3 version is under development). If you don’t want to use Python 2 (which should be easy enough using virtual environments), you’ll have to refer to the API documentation for how to do the curl calls. In these notes, I will use the command-line client. The installation should be as easy as:

pip install onecodex

Now we can try to classify the contents of our sample. In my case, the artisanal cheese metagenome file is called Sample2a.fastq.gz. We can query the One Codex API with gzipped files, so we don’t need to decompress it. First we need to be authenticated (at this point I am just following the tutorial here):

onecodex login

You will be prompted for your API key, which you’ll find under the Settings on the One Codex web site.

You can now list the available commands:

onecodex --help

which should show something like this:

usage: onecodex [-h] [--no-pretty-print] [--no-threads] [--max-threads N]
[--api-key API_KEY] [--version]
{upload,samples,analyses,references,logout,login} ...
One Codex Commands:
upload Upload one or more files to the One Codex platform
samples Retrieve uploaded samples
analyses Retrieve performed analyses
references Describe available Reference databses
logout Delete your API key (saved in ~/.onecodex)
login Add an API key (saved in ~/.onecodex)
One Codex Options:
-h, --help show this help message and exit
--no-pretty-print Do not pretty-print JSON responses
--no-threads Do not use multiple background threads to upload files
--max-threads N Specify a different max # of N upload threads
(defaults to 4)
--api-key API_KEY Manually provide a One Codex Beta API key
--version show program's version number and exit

Upload the sequences to the platform:

onecodex upload Sample2a.fastq.gz

This took me about five minutes – if you are using a small file like the 100-day infant gut metagenome it will be almost instantaneous. If we now give the following command:

onecodex analyses

it will show something similar to the following:

"analysis_status": "Pending",
"id": "6845bd3fa31c4c09",
"reference_id": "f5a3d51131104d7a",
"reference_name": "RefSeq 65 Complete Genomes",
"sample_filename": "Sample2a.fastq.gz",
"sample_id": "d4aff2bdf0db47cd"
"analysis_status": "Pending",
"id": "974c3ef01d254265",
"reference_id": "9a61796162d64790",
"reference_name": "One Codex 28K Database",
"sample_filename": "Sample2a.fastq.gz",
"sample_id": "d4aff2bdf0db47cd"

where the “analysis_status” of “Pending” indicates that the sample is still being processed. There are two entries because the sequences are being matched against two databases: the RefSeq 65 Complete Genomes and the One Codex 28K Database. According to the web site, “The RefSeq 65 Complete Genomes database […] includes 2718 bacterial genomes and 2318 viral genomes” and the “expanded One Codex 28k database includes the RefSeq 65 database as well as 22,710 additional genomes from the NCBI repository, for a total of 23,498 bacterial genomes, 3,995 viral genomes and 364 fungal genomes.”

After waiting for 10-15 minutes or so (due to some very recently added parallelization capabilities it should only take 4-5 minutes now), the “analysis_status” started showing “Success”. Now we can look at the results. Let’s check out the One Codex 28K database results. You just need to call onecodex analyses with the “id” value shown in one of the outputs above.

bmp:OneCodex mikaelhuss1$ onecodex analyses 974c3ef01d254265
"analysis_status": "Success",
"id": "974c3ef01d254265",
"n_reads": 2069638,
"p_mapped": 0.21960000000000002,
"reference_id": "9a61796162d64790",
"reference_name": "One Codex 28K Database",
"sample_filename": "Sample2a.fastq.gz",
"sample_id": "d4aff2bdf0db47cd",
"url": ""

So One Codex managed to assign a likely source organism to about 22% of the sequences. There is a URL to the results page. This URL is by default private to the user who created the analysis, but One Codex has recently added functionality to make results pages public if you want to share them, so I did that: Artisanal Cheese Metagenome Classification. Feel free to click around and explore the taxonomic tree and the other features.

You can also retrieve your analysis results as a JSON file:

onecodex analyses 974c3ef01d254265 --table > cheese.json

We see that the most abundantly detected bacterium in this artisanal cheese sample was Streptococcus macedonicus, which makes sense as that is a dairy isolate frequently found in fermented dairy products such as cheese.

Post Navigation