# NGS Workflows

June 8th 2016

Next generation sequencing. We all know that a fundamental practice in bioinformatics is the analysis of biological sequences. Similarities, functions, structures, associations, transcripts, proteins, RNA interference, regulation, interaction, DNA binding, the list goes on. Much can be hypothesized given some ATCGs (and some annotations).

However, its not plug and play. Various tools and algorithms exists for each step in NGS data pipelines. Each with their own advantages and disadvantages for a given set of data (e.g. bacterial vs. eukaryotic genomes). Their underlying algorithms can make assumptions which may not be true in all cases. New tools and methods are being developed and there are rarely adopted standards. Researchers today regularly construct hardcoded and unmaintanable scripts. I am not calling out these individuals on their coding practice, but rather positing that scripts without community maintained modular dependencies, with dependence on a specific environment configuration - let alone hardcoded absolute file references, are by their nature unfit for providing reproducible NGS workflows to the community at large. But hey, it

right?

A well written bash script can be version controlled, and dependencies can be described, however the consumer may not be able to achieve an identical environment. At the very least, it will be a painful setup process. Similarly, a python script is definitely more elegant and modular, but still suffers from issues such as pipeline reentrancy. One popular old tool is make, which can improve reentrancy by defining rules which have a target (output) and inputs. However, the syntax is not newcomer friendly and file pattern matching can be confusing or limited.

Not all hope is lost. There are have been many great efforts approaching this issue. One is snakemake which defines an elegant python-esque makefile with filename wildcarding, support for inline Python and R, and more. Another is nextflow, which goes a step further and describes pipelines through isolated (and containerizable) process blocks which communicate through channels. As well there are extras like galaxy, luigi, and bcbio.

In this blog post, I will define a simple variant calling pipeline. Then walk through the implementation of this pipeline using these four technologies:

1. bash
2. make
3. snakemake
4. nextflow

Then discuss other alternatives in brief. Finally I will propose where Bionode and JavaScript can fit into this ecosystem, and which specific issues can be addressed.

## Variant Calling Pipeline

We will be running a simple variant calling pipeline using a referenence genome and paired end genomic reads. For the sake of time when running the pipeline locally, we will use a small genome, Salmonella enterica, which has some paired end reads at about 100mb in size. With the reference genome at about 1.4mb, that provides about 70x coverage.

To follow along with this blog post, you will need to install: bionode-ncbi, sra-toolkit, bwa, samtools, bcftools, khmer, kmc, trimmomatic, snakemake, and nextflow.

Ha, I'm just kidding. Here is the Dockerfile: polyglot-ngs-01.

bionode-ncbi will be used to download the reads from the NCBI SRA, or sequence read archive. The reference could be downloaded with bionode-ncbi download assembly specie , but at the moment there is a bug where that downloads the rna_from_genomic rather than genomic file for some species. Once we have the an sra for Salmonella enterica, the next step is to generate the two fastq files for the two ended reads. Paired ends have a 5' $\rightarrow$ 3' set of reads and a 3' $\rightarrow$ 5' set of reads. This allows for much more confident alignment and is generally preferred over a single set of reads. For this we can use fastq-dump from sra tools: fastq-dump --split-files --skip-technical --gzip reads.sra This will produce reads_1.fastq.gz and reads_2.fastq.gz. fastq is essentially a fasta file that also includes quality scores. Quality scores are produced by the base calling methods employed by the given NGS machine. See the wikipedia pages for the fasta format and the fastq format. Filtering will be two step: 1. trim reads adapters with trimmomatic 2. filter out bad k-mers with a) khmer b) kmc This allows us to illustrate how much the pipeline tools let us swap in and out different tools for the same step (in this instance, khmer vs kmc) and then compare results. The filtering steps will complete by producing a reads.filtered.fastq.gz file. With other tools, it could have been a reads_1.filtered.fastq.gz and reads_2.fastq.gz. It depends whether or not one of the filtering tools creates an interleaved file holding both read directions. It does not matter too much, as the next step can handle both cases. If we wanted to skip filtering, we could just use reads_1.fastq.gz and reads_2.fastq.gz. Now we want to align the reads to the reference using bwa which is a Burrows-Wheeler transform based alignment tool. First, an FM-index needs to be constructed for the reference genome: bwa index reference.genomic.fna.gz Then we align using the reads, producing a sequence alignment map: bwa mem reference.genomic.fna.gz reads.filtered.fastq.gz > reads.sam We use samtools (also see wiki/SAMtools) to generate a binary alignment map: samtools view -bh reads.sam > reads.unsorted.bam and sort it: samtools sort reads.unsorted.bam -o reads.bam We could have piped the last three steps together, avoiding file writing/reading overhead: bwa mem ref reads | samtools view -bh - | samtools sort - -o reads.bam and then index it (creates reads.bam.bai - binary alignment index): samtools index reads.bam At this point, we have everything we need to call variants: samtools mpileup -uf reference.genomic.fna reads.bam | \ bcftools call -c - > reads.vcf mpileup creates a BCF pileup file describing the base calls of aligned reads. Then bcftools call takes this and generates a variant call format file. Since samtools was complaining about the reference.genomic.fna.gz that comes from the NCBI Assemblies database - something to do with the compression format, I first decompressed it with: bgzip -d reference.genomic.fna.gz Thats it! Thats how to get from SRA $\rightarrow$ VCF using bionode, sra tools, trimming tools, filtering tools, bwa, samtools, and bcftools. The next sections will go over how to improve the reproducibility, reentrancy, ease of development, etc. of this workflow. ## Topics For each tool, we will inspect the following topics. ### Basic Structure • quick intro to the tool, how it is written ### Iterative Development • reentrancy • error debugging ### Metrics • time for each task • resource usage • reports ### Scaling • extend to many species • run on a cluster/cloud system • distribute/reproducibility ## bash The first obvious tool to use constructing a pipeline composed of a list of shell commands is a simple bash script. Essentially, we can take the commands from above and arrange them in a linear manner. You can see the full bash pipeline here: basic-snp-calling.sh. ### Basic Structure The above section introduced the tools and their commands required to run a simple variant calling pipeline. However, all of the file names were hardcoded: reference.genomic.fna.gz, reads.fastq.gz, etc. In a real analysis, we would like to generalize so that we can provide paramaters defining the specie and reads to use. Then, for example, we can run the pipeline over many species and inspect related hypotheses. Within a bash script we can attempt to carry this out using environment variables. Depending on how these variables are defined, they may be accessible from different places. For example, the PATH variable (try echoPATH) in your shell is available all the time. This is because it was defined using export. Without export, a variable is only available within in its current context. Consider:

main.sh

#!/bin/bash
READS_ID=2492428 # no spaces around =!
./downloadReads.sh

#!/bin/bash
bionode-ncbi download sra $READS_ID In this case, READS_ID will evaluate to blank. Alternatively you need to export READS_ID=2492428 to have the variable available in other contexts. This is important to make note of, because complex shell scripts will likely be split across multiple files, in which case it is important to understand when and when not to use export. Blindly using export everywhere is not recommended. With an understanding of environment variables out of the way, we can use them to define some initial settings for the script: # Salmonella enterica REFERENCE_NAME='GCA_000006945.2_ASM694v2' REFERENCE="${REFERENCE_NAME}_genomic.fna.gz"
REFERENCE_URL="http://ftp.ncbi.nlm.nih.gov/genomes/all/$REFERENCE_NAME/$REFERENCE"
FILTER_MODE='khmer' # 'kmc', 'none'

READSFQ is defined with export so it can be available from the filter scripts which have been separated out from the main file. Then files used as input and output can be described with these variables:

echo "START fastq-dump"
fastq-dump --split-files --skip-technical --gzip $READS/**.sra echo "END fastq-dump" I also notify the start and end of each task, manually. This is cumbersome and cluttering, and adding more useful features like time taken would be even more messy. Up until the filtering, the pipeline is completely linear. However, we would like to filter using two tools: khmer and kmc. One is significantly faster, but may produce more erroneous results. But that does not mean its useless, it can be useful to favour speed and number of species over specificity at the expense of less trials. Ideally, the swap from fast to slow should be a simple swap. As well, it can be useful to compare where exactly the two methods differ in their results. Enter the if [ condtion ]; then … fi block: if [$FILTER_MODE == 'kmc' ]; then
if [ ! -d $TMPDIR ]; then mkdir -p$TMPDIR
fi
echo "START filtering with kmc"
export READS_TO_ALIGN="$READSFQ.trim.pe.kmc.fastq.gz" ./filter_kmc.sh echo "END filtering with kmc" elif [$FILTER_MODE == 'khmer' ]; then
echo "Using khmer mode"
export READS_TO_ALIGN="$READSFQ.trim.pe.khmer.fastq.gz" ./filter_khmer.sh FINAL_OUTPUT="${READS}-khmer.vcf"
else
@echo "=== Running $@ ===" @export start=date +%s; \ bwa mem -t${THREADS} reference.genomic.fna.gz ${READS_1}${READS_2} > $@; \ export end=date +%s; \ export runtime=$$(($$end-$$start)); \ echo "Target @ took$$runtime seconds" ### Iterative Development Iterative development in make is much improved over a bash script. This is because you can call a specific rule at invocation: make reads.sam for example. As well, it is common practice to include a clean rule. It is possible in make to define targets that don't actually create the target file. Since the target is not actually created (even though the command in that rule has ran and worked as expected), since make skips rules if the target already exists, it will unnecessarily rerun the rule. My general workflow writing a make pipeline was to update the prequisite of rule all as new rules were added, and each task from before would be skipped. One way to get around this issue is to create "flag files": trim.happened: fastq-dump.log @echo "=== Running$@ ==="
@export start=date +%s; \
java -jar ${TRIMMOMATIC} PE -phred33 \ reads_1.fastq.gz reads_2.fastq.gz \ reads_1.trim.pe.fastq.gz reads_1.trim.se.fastq.gz \ reads_2.trim.pe.fastq.gz reads_2.trim.se.fastq.gz \ ILLUMINACLIP:${ADAPTERS}/TruSeq3-PE.fa:2:30:10 \
LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:36 > $@; \ export end=date +%s; \ export runtime=$$(($$end-$$start)); \ cat @; \ echo "Target @ took$$runtime seconds" Here, the stdout of trimmomatic is sent to trim.happened. Even if there is no stdout, an empty file will get made, and then the next run this rule can be skipped. ### Metrics To get time spent metrics, I wrapped each command in a simple delta time calculation: time: @echo "=== Running$@ ==="
@export start=date +%s; \
sleep 2; \
export end=date +%s; \
export runtime=$$(($$end-$$start)); \ echo "Target @ took$$runtime seconds"

This clutters the code fast, and is unmaintainable. It might be possible to do it cleaner, but I did not spend time trying obscure solutions. The point is that there is no "out of the box" way to get task metrics with make. You can imagine how tricky it might be to also get RAM/CPU usage for each task.

### Scaling

Scaling with make is also difficult. There is no easy way to expand the pipeline to multiple species, and it is difficulty to "variabilize" outputs: targets need to strings.

## snakemake

The Makefile was much more organized than the bash script, but still of "a low level".

Snakemake was a refreshing take on the make style, but with many more features and powered by a high level language. Including Python integration everywhere, tasks to script in Python, Bash, and R, and some metrics out of the box. As well as some neat wildcarding. The complete Snakefile is here.

### Basic Structure

You start a Snakemake workflow very similar to a Makefile, defining a global rule and what you want it to create:

rule all:
input: FINAL_FILES

So what is FINAL_FILES? Since Snakemake has direct Python integration, we can actually compute it as a local variable. Heres the header for the Snakefile just before

rule all:

species = {
'Salmonella-enterica': {
'reference_url': 'http://ftp.ncbi.nlm.nih.gov/genomes/all/GCA_000988525.2_ASM98852v2/GCA_000988525.2_ASM98852v2_genomic.fna.gz'
}
}

TEMP='./tmp'

FINAL_FILES = [specie+'.vcf' for specie in species]

This will end up with FINAL_FILES being ['Salmonella-enterica.vcf']. See this rule which will be one of the first in the executed pipeline (here I hardcoded the specie name to better illustrate):

rule all:
input: 'Salmonella-enterica.vcf'

output: '{specie}.sra'
run:
shell('''
''')

rule call:
input: '{specie}.sra'
output: '{specie}.vcf'
shell: 'magic {input} > {output}'

The first rule will be triggered and will be looking for another rule that has Salmonella-enterica.vcf in its output. It won't find exactly that, but because of Snakemake's wildcarding, {specie}.vcf will do (from rule call), and then within the call rule, the value of wildcards.specie will be Salmonella-enterica. Then it will move onto download_sra. This example also illustrates how you can use run and shell() to mix Python and shell code. You can also use script to run a Python or R script, and use the R() function to execute R code in a rule.

Another neat feature with Snakemake is the ability to drop in "wrappers":

rule bwa_mem:
input:
ref = '{specie}.genomic.fna.gz',
sample = ['{specie}_'+num+'.fastq.gz' for num in ['1', '2']],
index_files = ['{specie}.genomic.fna.gz.'+suffix for suffix in ['amb', 'ann', 'bwt', 'pac', 'sa']]
log: 'logs/bwa_mem/{specie}.log'
output: '{specie}.sam'
wrapper: '0.0.8/bio/bwa_mem'

These wrappers come from the wrappers repository. They run predefined commands using specific input variables. While this is great, there is some overhead in having to check the source to see what is actually happening, and by extension, you then need an internet connection to do so. There does not seem to be a huge list of wrappers, it is too bad it does not seem to have caught on extensively.

Still have the problem of using log/flag files for task dependency, but less so, since you can define "custom" outputs, also has touch(flag) built in

Could not figure out how to ecomical branching, wildcard regexes create ambiguity:

Here it unnecessarily redoes certain steps. This is because Salmonella-enterica.trim.vcf and Salmonella-enterica.trim.vcf both match {specie}.vcf. At first, Snakemake complained about ambiguous rules due to this conflict. I was able to generate the DAG above by using custom regexes for the wildcard in a few rules: {specie,[a-zA-z-]+}. However, since this then creates one set of rules with wildcard.specie = Salmonella-enterica and another with wildcard.specie = Salmonella-enterica.trim, and then that wildcard moves all the way to the first rule, and basically two pipelines are ran. I was not able to figure out how optimize this using Snakemake - if anyone can, let me know!

Moreover - because the pull workflow style depends on targets and prerequisites, it can be difficult to achieve a branch-merge pipeline. We will see with Nextflow, which follows the dataflow paradigm, or push, it is natural to describe such pipelines.

For a more complicated (and real world) snakemake setup, take a look at pachterlab/kallisto_paper_analysis/Snakefile.

### Iterative Development

Iterative development in Snakemake is about the same as with make. With wildcarding, the clean rule can be made more specific, for example:

rule clean:
input: '{specie}.vcf'
shell: '''
rm {specie}.sra {specie}.bam;
'''

As opposed to rm *.sra *.bam.

### Metrics

You can opt in to store rule benchmarks and logs:

rule call:
input:
bam = '{specie}.bam',
alignment_index = '{specie}.bam.bai',
reference = '{specie}.genomic.fna'
output: '{specie}.vcf'
log: 'logs/call/{specie}.log'
benchmark: 'benchmarks/call/{specie}.txt'
shell: 'samtools mpileup -uf {input.reference} {input.bam} | bcftools call -c - > {output}'

However, you will need to make sure you name these files appropiately to avoid overwriting. An example benchmark file looks like:

s    h:m:s
30.32477617263794    0:00:30.324776

There is no indication of which task the benchmark is for, meaning if you want a comprehensive report of your pipeline you will need to write a script that concatenates the benchmarks in the correct order.

With graphviz installed, snakemake can create a plot of the DAG for your pipeline:

snakemake --dag | dot -Tpng > dag.png

You can see an example of this above.

### Scaling

With Snakemake it is easy to scale to multiple species. In my Snakefile, I simply just add more keys to the species dictionary. Since each output file is prefixed by the species name, there will be no file overlaps. You need to be vigilant with output names and ensure yourself there will be no conflicts.

You can specify clustering configuration by writing a cluster.config file and using its values in the call to Snakemake. The example from the documentation:

Snakefile

rule all:
input: "input1.txt", "input2.txt"

rule compute1:
output: "input1.txt"
shell: "touch input1.txt"

rule compute2:
output: "input2.txt"
shell: "touch input2.txt"

cluster.json

{
"__default__" :
{
"account" : "my account",
"time" : "00:15:00",
"n" : 1,
"partition" : "core"
},
"compute1" :
{
"time" : "00:20:00"
}
}

And the call to Snakemake:

snakemake -j 999 --cluster-config cluster.json --cluster "sbatch -A {cluster.account} -p {cluster.partition} -n {cluster.n}  -t {cluster.time}"

While nextflow is providing a simple config based way to pass params into your clustering command, the actual command: sbatch is written manually. We will say with Nextflow, even this aspect of clustering is abstracted away (into "executor"). See also job properties to see how to write a generic job wrapper for clustering.

## nextflow

Nextflow is a more recent tool, and approaches the workflow problem in a push sense. Similar to how Snakemake allows direct Python integration, Nextflow uses Groovy. While Snakemake let you write rules with inline Python, R, and shell, in Nextflow, you can use any scripting language. See main.nf for the complete pipeline.

Nextflow follows the dataflow programming paradigm (push) which is also called stream processing or reactive programming. While in Snakemake you define rule all's prerequisites, and then "work backwards" from there (pull), in the dataflow model you can think and write in the order that tasks will happen. Since the push model does not depend on inferring a complete dependency tree at the initialization, you can introduce dynamic structures into the pipeline. For example, choosing between two tasks to run based on the output of a previous task. One fallback with the push model is that since it can be dynamic, it is difficult to then perform a "dry run", or generate a DAG ahead of time.

### Basic Structure

Nextflow uses processes as rules, and each process will occur in its own folder in /work. Since each process takes place in its own folder, using output from one task as input in another cannot be done the "conventional way". But that is alright, because Nextflow provides channels to communicate between processes.

Consider:

#!/usr/bin/env nextflow

species = [
'Salmonella-enterica': [
'referenceURL': 'http://ftp.ncbi.nlm.nih.gov/genomes/all/GCA_000988525.2_ASM98852v2/GCA_000988525.2_ASM98852v2_genomic.fna.gz',
]
]

container 'bionode/bionode-ncbi'

"""
bionode-ncbi download sra $readsID > tmp """ } process extractSRA { container 'inutano/sra-toolkit' input: file read from reads output: file '*.fastq.gz' into samples """ fastq-dump --split-files --skip-technical --gzip$read
"""
}

See the documentation on [processes] for more. As well, there are more types of channels than val and file, see [channels]. If you are new to [Groovy], and species.collect { it.value.readsID } confuses you, think of it as species.map(specie => specie.value.readsID). It is important to note that while channels may appear confusing at first (why not just use filenames?), they are an elegant solution to a problem we had with Snakemake: file name overlap. With Nextflow, you can be guarenteed no files will ever overwrite each other (unless you do so yourself in one process). As well, this abstracts away the filename, making commands appear generalized:

process indexReference {
container 'biodckr/bwa'

input: file reference from referenceGenomeGz2
output: file '*.gz.*' into referenceIndexes

"""
bwa index $reference """ } There is no mental overhead from managing {specie}.genomic.fna.gz, instead we can simply use the variable $reference. This makes the commands in our processes easy to read and comprehend. We can even "hardcode" output file names in an extremely general way without any fear of file overlap:

process decompressReference {
container 'biodckrdev/htslib'

input: file referenceGenome from referenceGenomeGz1
output: file 'reference.genomic.fna' into referenceGenomes

"""
bgzip -d $referenceGenome --stdout > reference.genomic.fna """ } Here we output to reference.genomic.fna. A filename that would surely cause problems in any other worflow system. Something else to discuss with Nextflow is how to handle a channel being consumed by multiple processes. Since a channel is a FIFO queue in Nextflow, once one process uses it, it is "emptied" and cannot be consumed by another process. The way to get around this is to create the original channel called myOutput and then "clone" it via into $n$ channels to be consumed by $n$ processes: process downloadReference { container true input: val referenceURL from species.collect { it.value.referenceURL } output: file 'reference.genomic.fna.gz' into referenceGenomeGz """ appropriate/curl$referenceURL -o reference.genomic.fna.gz
"""
}

// fork reference genome into three other channels
( referenceGenomeGz1,
referenceGenomeGz2,
referenceGenomeGz3 ) = referenceGenomeGz.into(3)

This is also an example of an executable container, where the Dockerfile ends in CMD ["curl"].

However, I encountered some issues when trying to dockerize everything. TODO talk about dockerizing processes in a pipe.

Finally, wherease Snakemake only allowed Python and R inside rules, with Nextflow you can use any scripting language.

### Iterative Development

Iterative developmen with Nextflow is much improved over the other tools. I felt as though I could extend the pipeline, adding new processes, without having to worry about updating a rule all rule, or cleaning up old files. Nextflow seamlessly recognizes cached process' results when given the -resume flag.

Debugging errors in processes works great. When an error occurs, you can cd into the relevant /work directory, and Nextflow provides log files, and a file that describes which command exactly was ran. This allows you to debug the error in the same environment as it will be ran, and then resume the workflow from that point.

### Metrics

Nextflow can create timeline charts:

and a DAG diagram of the pipeline (also notice I was able to create a "forking" pipeline unlike with Snakemake):

### Scaling

You might have noticed the container directive in the above processes. Nextflow comes with built-in docker integration, which is great. Each process can run in its own container. This helps improve the portability and reproducibiltiy of the workflow. All a consumer needs installed on their system is Docker and Nextflow (and you can even run Nextflow in Docker). Each container can use a tagged version, ensuring when someone else runs the pipeline, they are using the exact same version of each tool. You could, of course use Docker in your Snakemake commands, but there would be overhead from volume mounting (mapping a local folder to a folder inside the container); Nextflow handles all that for you. As well, Nextflow has built in support for many cluster engines, which can be enabled by defining the executor in nextflow.config. Another feature to not is Nextflow's integration with GitHub. With a main.nf in your repo, you can run a pipeline with nextflow run username/repo.

Finally, be sure to check out awesome-nextflow!

## others

awesome-pipeline is a great list of many pipeline and workflow tools. Here are some I think are more relevant, and a few extras (though you should try to look through all of them, at least look at these):

• anduril - Component-based workflow framework for scientific data analysis
• antha - High-level language for biology
• bpipe - Tool for running and managing bioinformatics pipelines
• Conan2 - Light-weight workflow management application
• cosmos - Python library for massively parallel workflows
• drake - Robust DSL akin to Make, implemented in Clojure
• kronos - Workflow assembler for cancer genome analytics and informatics
• loom - Tool for running bioinformatics workflows locally or in the cloud
• moa - Lightweight workflows in bioinformatics
• OpenGE - Accelerated framework for manipulating and interpreting high-throughput sequencing data
• pipengine - Ruby based launcher for complex biological pipelines
• ruffus - Computation Pipeline library for Python
• YAP - Extensible parallel framework, written in Python using OpenMPI libraries
• clusterflow - Command-line tool which uses common cluster managers to run bioinformatics pipelines
• hive - System for creating and running pipelines on a distributed compute resource
• google cloud dataflow - unified programming model and a managed service for developing and executing a wide range of data processing patterns including ETL, batch computation, and continuous computation
• makeflow - Workflow engine for executing large complex workflows on clusters
• scoop - Scalable Concurrent Operations in Python
• luigi and sciluigi - Python module that helps you build complex pipelines of batch jobs, luigi wrapper for scientific workflows
• galaxy - Web-based platform for biomedical research
• taverna - Domain independent workflow system
• rabix - implementation of CWL2
• cuneiform - Advanced functional workflow language and framework, implemented in Erlang
• mario - Scala library for defining data pipelines
• airflow - Python-based workflow system created by AirBnb
• pinball - Python based workflow engine by Pinterest

Quite a lot.

## Conclusion

On a scale from 1-5, these are my ratings for each tool. Mostly as relative to each other, rather than absolutely.

Tool Structure Iterative Dev. Metrics Scale Reproducibility
bash 1 1 1 1 1
make 2 2 1 1 1
Snakemake 4 3 3 3 4
Nextflow 4 5 5 5 5

I have not investigated the "others" from above to warrant their ranking.

A quick summary:

bash

• "generic" scripting
• simple variable interpolation - $foo • no metrics, difficult to scale • no reentrancy, poor iterative development make • structure more pipeline oriented than bash - tasks with input (prerequisites) and output (target) • introduces some element of reentrancy by skipping rules whose target already exists = improved iterative development over bash • can "fork" by defining variables that change a rule's prerequisites, and running again • no metrics, difficult to scale Snakemake • enhanced scripting over bash/make with Python while using make's well known rules structure • improved {input}, {output} syntax and power (e.g. {input.a} or {input[n]}) over make • run Python, shell, or R in rule • enhanced wildcarding: easier to understand, more powerful than make's % • benchmarks are OK - need to do manual work to get a full pipeline report • DAG is nice • dry run is nice (and possible because of "pull" paradigm) • can scale to multiple species easily, just need to make sure file names do not overlap • cannot create forking paths (e.g. use same reference for two different filtering tools). Could do it by changing FINAL_OUTPUT and running again, but then thats basically the same as it was in make. (again, if you know how to do this without wildcarding ambiguity - let me know!) Nextflow • dataflow paradigm - "push" • as a consequence of push, no dry run • as a consequence of push, flexible forking and dynamic pipelines • scale to multiple species effortlessly with no overlapping file name concerns • timeline, DAG, benchmarks metrics are all very nice • Docker is nice - improves reproducibility and shareability • cannot handle large stdout | stdin pipes across channels (e.g. one process for bwa mem and another for samtools view) bionode-waterwheel proposal • no DSL to learn - just JavaScript. Functional, built around async, events, and streams. • streams resonate well with push paradigm • support for piping stdin, stdout around, e.g. bwa.mem().pipe(samtools.view()) . This enhances modularity of pipeline, rather than having one rule or process doing bwa mem | samtools view, which is really two commands, each deserving their own input, output, parameter definitions. This can aid in improving reproducibility and modularity, as tools with defined input, output, params could be confidentally dropped into the pipeline. • moduler interoperation with web apps, native apps (Electron), services (Slack, email). Imagine pipeline logs being sent over a websocket to your browser. • interoperation with npm ecosystem • integrate with CWL spec • metrics will be easier to consume since output will be JSON to be consumed by d3, browser apps, etc • modular, specific, customizable waterwheel "backend" can be integrated into browser or native apps, bringing more power to pipeline GUIs than Galaxy for instance, where it is complicated to develop a custom pipeline (some labs hire someone to create a custom xml config for their specific pipeline so that the wet lab can "click to play" in Galaxy) • variable interpolation - like ES6 template literals, which can take any valid JS expression: const PORT = config.PORT const template = Server listening on port${PORT}

which can actually evaluate any JS expression:

[1, 2, 3, 4].map(num => is even: ${num % 2 === 0 ? 'true' : 'false'}) • simple examples with small datasets can be browser compatibile (see: nbind). Live browser examples are great for education - "run your own NGS pipeline, from the browser" • how to handle interative development is an open question. Can fork all streams into files for reentrancy while in a "develop" mode. A draft pipeline with bionode-waterwheel: const ncbi = require('bionode-ncbi') const wrapper = require('bionode-wrapper') const waterwheel = require('bionode-waterwheel') const { task, join, run } = waterwheel const { stdout, stdin, file, directory } = waterwheel.types // Can be passed in from CLI // params for final pipeline call. params are things that do not // change how items are passed between processes, but decide output for the // pipeline as a whole. For example, species name and reads accession. const pipelineParams = { specie: 'Salmonella-enterica', readsID: '2492428', output: 'Salmonella-enterica.vcf' } const sra = task({ output: stdout() }, ncbi.download('sra', '{params.readsID}')) const reference = task({ output: stdout() }, ncbi.download('assembly', '{params.specie}')) const bwa_index = task({ input: file(), output: file() }, wrapper('bwa index {input}')) // some tools you cannot decide output file // so, tell waterwheel to stream a file as stdout // there is --stdout for fastq-dump to give a streamed fastq, but trimmomatic // wants reads_1 and reads_2 // tools that are not bionode will need to wrapper()ed const extract = task({ input: file('reads.sra'), output: file([1, 2].map(n => reads_${n}.fastq.gz))
}, wrapper('fastq-dump --split-files --skip-technical --gzip {input}'))

input: file([1, 2].map(n => reads_${n}.fastq.gz)), output: { pe: file([1, 2].map(n => reads_${n}.trim.pe.fastq.gz),
se: file([1, 2].map(n => reads_${n}.trim.se.fastq.gz) }, opts: { adapters: '../adapters' } }, wrapper(''' trimmomatic PE -phred33 \ {input[0]} {input[1]} \ {output.pe[0]} {output.se[0]} \ {output.pe[1]} {output.se[1]} \ ILLUMINACLIP:{opts.adapters}/TruSeq3-PE.fa:2:30:10 \ LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:36 \ ''')) const merge = task({ input: file(), output: stdout() }, wrapper('seqtk mergepe {input}')) const gzip = task({ input: stdin(), output: stdout() }, wrapper('gzip - > {output}')) // Branching: these two filtering types produce the same type of output // So we can pass an array, and define them both under the same input/output const filter = task({ input: file() output: file() opts: { tmpDir: directory() } }, [wrapper(''' kmc -k{params.KMERSIZE} -m{params.MEMORYGB} -t{params.THREADS} {input} reads.trim.pe.kmc {opts.tmp} \ kmc_tools filter reads.trim.pe.kmc -cx{params.MINCOVERAGE} {input} -ci0 -cx0 {output} \ '''), wrapper(''' load-into-counting.py -N 4 -k {params.KMERSIZE} -M {params.MEMORYGB}e9 -T {params.THREADS} reads.trim.pe.fastq.gz.kh {input} \ abundance-dist.py reads.trim.pe.fastq.gz.kh {input} reads.trim.pe.fastq.gz.kh.hist \ filter-abund.py -T {params.THREADS} -C${MINCOVERAGE} reads.trim.pe.fastq.gz.kh -o {output} {input} \
''')])

// file() dependencies will stop stream.
// for example, need to wait on an index file to be made before aligning
input: {
reference: file(),
index: file(), // check for reference indexing
sample: file() // output of filter
}
output: stdout()
}, wrapper('bwa mem {input.reference} {input.sample}'))

// This is where streams in Node can really show
// In snakemake or Nextflow, this would be bwa mem | samtools view
// Which is less modular, reproducible, containerizable
input: stdin(),
output: stdout()
}, wrapper('samtools view -Sbh {input}'))

input: stdin(),
output: stdout()
}, wrapper('samtools sort {input}'))

input: stdin(),
output: stdout()
}, wrapper('samtools index {input}'))

// these will all get piped through each other:
// bwa_mem().pipe(samtools_view()).pipe(samtools_sort()).pipe(samtools_index())
const align = join([bwa_mem, samtools_view, samtools_sort, samtools_index])

input: {
bam: file(),
index: file(),
reference: file()
},
output: stdout()
}, wrapper('samtools mpileup -uf {input.bam} {input.reference}'))

input: stdin(),
output: stdout()
}, wrapper('bcftools call -c {input}'))

const callVariants = join([samtools_mpileup, bcftools_call])

// need a way to use output of another task as input for this one
const pipeline = join(
[sra, extract, trim, merge],
// this creates a branching
filter,
align,
callVariants
)

// Run the whole pipeline, passing in params
run(pipelineParams, pipeline).pipe(task(fs.createWriteStream('{output}')))