The aim of this tutorial is twofold:

1. It introduces general patterns of working with high-throughput sequencing data in Galaxy,
2. It uses SARS-CoV-2 sequencing data analysis as a concrete example of how to identify genomic mutations from high-throughput sequencing data for a batch of viral isolates in Galaxy.

Agenda

In this tutorial, we will cover:

1. Prepare analysis history and data
   1. Create a new history for this analysis
   2. Find, obtain and filter information about public sequencing data
   3. Retrieve selected sequencing data from the SRA
   4. Get the reference genome data
2. Identification of mutations in the batch of SARS-Cov-2 sequencing data
   1. Adapter trimming with fastp
   2. Alignment with Map with BWA-MEM
   3. Remove duplicates with MarkDuplicates
   4. Generate alignment statistics with Samtools stats
   5. Realign reads with lofreq viterbi
   6. Add indel qualities with lofreq Insert indel qualities
   7. Call Variants using lofreq Call variants
   8. Annotate variant effects with SnpEff eff:
   9. Create table of variants using SnpSift Extract Fields
   10. Summarize data with MultiQC
3. Conclusion

Prepare analysis history and data

Create a new history for this analysis

Any analysis should get its own Galaxy history. So let’s start by creating a new one:

Hands-on: Prepare the Galaxy history

1. Create a new history for this analysis

   Tip: Creating a new history

   To create a new history simply click the new-history icon at the top of the history panel:

   

2. Rename the history

   Tip: Renaming a history

   1. Click on galaxy-pencil (Edit) next to the history name (which by default is “Unnamed history”)
   2. Type the new name: SARS-CoV-2 sequence data analysis
   3. Click on Save
   4. To cancel renaming, click the galaxy-undo “Cancel” button

   If you do not have the galaxy-pencil (Edit) next to the history name (which can be the case if you are using an older version of Galaxy) do the following:

   1. Click on Unnamed history (or the current name of the history) (Click to rename history) at the top of your history panel
   2. Type the new name: SARS-CoV-2 sequence data analysis
   3. Press Enter

Find, obtain and filter information about public sequencing data

First we need to find some good, public datasets to play with.

The Sequence Read Archive (SRA) is the primary archive of unassembled reads operated by the US National Institutes of Health (NIH). SRA is a great place to get the sequencing data that underlie publications and studies. The study we are interested in for this tutorial is one on sequencing of SARS-CoV-2 viral isolates that were obtained in the Boston area in the first months of the COVID-19 pandemic (Lemieux et al. 2021).

So lets see how we can find its raw data in the SRA, and how we can get some of that data into Galaxy to analyze it. The Acknowledgments section of the publication mentions that raw reads from the study have been deposited in the SRA under BioProject PRJNA622837 so that’s going to be our starting point.

Hands-on: Find the example data in the SRA

1. Go to NCBI’s SRA page by pointing your browser to https://www.ncbi.nlm.nih.gov/sra

2. In the search box enter PRJNA622837[BioProject]

   Oh, this finds a lot of samples (more than 22,000 at the time of writing)! This is because the BioProject ID we used is that of “SARS-CoV-2 Patient Sequencing from the Broad Institute”, which has seen many more sample submissions since the published study. Let’s refine our search a bit.

3. Replace your previous search with PRJNA622837[BioProject] AND RNA-Seq[Strategy]

   This returns only those samples from the BioProject, for which the sequencing strategy was RNA-Seq.

   At the time of writing, 1908 hits are retained with this query, which is still more than the approximately 800 genomes mentioned in the publication, but we got a lot closer.

   Comment

   The vast majority of what has been excluded by adding RNA-Seq[Strategy] to the query are datasets for which the “Strategy” is AMPLICON. For these datasets, the viral RNA (or more precisely, the cDNA obtained from it) got amplified with a tiled-amplicon protocol. While tiled-amplicon sequencing has emerged as a standard way of sequencing SARS-CoV-2 samples over the course of the pandemic, the corresponding protocols/primer schemes were simply not available during the early phase of the pandemic.

4. Instead of inspecting all 1908 hits one-by-one through the web interface, lets download the metadata for all samples in a single file
   1. Click on the Send to: dropdown at the top of the search results list
   2. Under Choose Destination, select Run Selector
   3. Click Go

   

   You will be taken to the SRA Run Selector page, where you can browse all of the metadata of all search hits rather conveniently.

5. In the Select table, click Metadata in the Download column

   

   This will download the metadata for all retrieved sequencing runs as a comma-separated text file.

Now that we have downloaded this file we can go to a Galaxy instance and start processing it.

Comment

Note that the file we just downloaded is not sequencing data itself. Rather, it is metadata describing the data itself. We will use a few Galaxy tools to filter this list down to just a few accessions that will be used in the remainder of this tutorial.

Hands-on: Upload the metadata file into Galaxy

1. In Galaxy, click galaxy-upload Upload Data

   

2. In the Upload dialog box, click Choose local files

   

3. Find and select the downloaded metadata file on your computer
4. Click Start
5. Close the dialog by pressing Close

6. Once the dataset has finished uploading to your history, you can galaxy-eye inspect its contents.

   You will see that this file contains the same metadata that you could preview on the SRA Run Selector page.

In Galaxy, it is rather easy to perform further filtering of the metadata records. For example, the publication mentions that the authors analyzed samples collected between 4 March and 9 May 2020.

If you inspect the metadata, you should see a column Collection_Date. That looks useful!

Hands-on: Filter metadata lines by collection date range

1. Filter data on any column using simple expressions

   • param-file “Filter”: the uploaded metadata

   • “With following condition”: "2020-03-04" <= c11 <= "2020-05-09"

     Column 11 (c11) is the Collection_Date column in this case. If your input has this column in a different position, you would need to adjust the filter condition above accordingly.

   • “Number of header lines to skip”: 1

So now we are down to sequencing runs from the same date range as studied in the publication - though we still have more runs than samples discussed in the publication. Note, however, that the publication talks about assembled genomes, and it is possible that some of the sequencing runs listed in our metadata do not contain good enough data to assemble any SARS-CoV-2 genome information, or it could be that some viral samples got sequenced in several sequencing runs to obtain enough data.

We could explore this situation in more detail, and Galaxy could also process all the sequencing runs we are currently left with, but for the sake of simplicity of the tutorial, we want to select a much smaller subset of sequencing runs now anyway. In particular, from previous experience with this data we suggest to continue with just the following two interesting datasets: SRR11954102 and SRR12733957. So let’s pull their identifiers out of the metadata file.

Comment

The following steps may seem a bit silly given that we just revealed the two identifiers above. However, think of them as placeholders for extracting run identifiers based on some other criteria.

Hands-on: Extracting a subset of identifiers

1. Select lines that match an expression

   • param-file “Select lines from”: the metadata filter by collection date; output of Filter tool
   • “that”: Matching
   • “the pattern”: SRR12733957|SRR11954102

2. Advanced Cut ( Galaxy version 1.1.0)

   • param-file “File to cut”: the metadata with only two runs retained; output of Select tool
   • Under “Cut by”
     • “List of Fields”: Column: 1

   Warning: Beware of Cuts

   The section below uses Cut tool. There are two cut tools in Galaxy due to historical reasons. This example uses tool with the full name Cut columns from a table (cut). However, the same logic applies to the other tool. It simply has a slightly different interface.

At this point, you should have a dataset with just two lines and a single column with this content:

SRR12733957
SRR11954102

Retrieve selected sequencing data from the SRA

Now that we have our identifiers of interest extracted, we are ready to download the actual sequencing data. Since this is a very common need Galaxy offers a dedicated tool for the purpose of downloading sequencing data from the SRA identified via its run accession.

Hands-on: Retrieve sequencing data from SRA via run accessions

1. Faster Download and Extract Reads in FASTQ ( Galaxy version 3.0.3+galaxy0) with the following parameters:
   • “select input type”: List of SRA accession, one per line
     • param-file “sra accession list”: the dataset with the two run accessions; output of Cut

Several entries should have been created in your history panel when you submitted the previous job:

• Pair-end data (fasterq-dump): Contains Paired-end datasets (if available)
• Single-end data (fasterq-dump) Contains Single-end datasets (if available)
• Other data (fasterq-dump) Contains Unpaired datasets (if available)
• fasterq-dump log Contains Information about the tool execution

The first three items are actually collections of datasets.

When the tool run is finished (all history items created by it are green / done), take a moment to explore the collections by first clicking on the collection name in the history panel. This takes you inside the collection and shows you the datasets in it. You can then navigate back to the outer level of your history.

You should find that only the Pair-end data collection contains any sequencing data, which makes sense as both samples have been paired-end sequenced on the Illumina platform.

Get the reference genome data

Since our data analysis will involve mapping the sequenced reads just downloaded to the SARS-CoV-2 reference, we also need to obtain that reference now.

Hands-on: Get the reference genome data

1. Import the following file into your history:

   https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/009/858/895/GCF_009858895.2_ASM985889v3/GCF_009858895.2_ASM985889v3_genomic.fna.gz
   

   Tip: Importing via links

   • Copy the link location

   • Click galaxy-upload Upload Data at the top of the tool panel

   • Select galaxy-wf-edit Paste/Fetch Data

   • Paste the link(s) into the text field

   • Press Start

   • Close the window

Identification of mutations in the batch of SARS-Cov-2 sequencing data

As our actual analysis we will now perform all steps necessary to identify mutations present in the genomes of our minimal batch of two samples with respect to the SARS-CoV-2 Wuhan-Hu-1 reference sequence.

We will perform quality control and read trimming, map the reads from both samples to the reference, and perform variant calling. Finally we will annotate each sample’s variants with their predicted functional impact and report our findings in a table.

We will also summarize different quality metrics obtained throughout the analysis in one nice summary report.

Adapter trimming with fastp

Removing sequencing adapters improves alignments and variant calling. fastp tool can automatically detect widely used sequencing adapters.

Hands-on: Task description

1. fastp tool with the following parameters:
   • “Single-end or paired reads”: Paired Collection
     • param-file “Select paired collection(s)”: list_paired (output of Faster Download and Extract Reads in FASTQ tool)
   • In “Output Options”:
     • “Output JSON report”: Yes

Alignment with Map with BWA-MEM

BWA-MEM tool is a widely used sequence aligner for short-read sequencing datasets such as those we are analysing in this tutorial.

Hands-on: Align sequencing reads to reference genome

1. Map with BWA-MEM tool with the following parameters:
   • “Will you select a reference genome from your history or use a built-in index?”: Use a genome from history and build index
     • param-file “Use the following dataset as the reference sequence”: output (Input dataset)
   • “Single or Paired-end reads”: Paired Collection
     • param-file “Select a paired collection”: output_paired_coll (output of fastp tool)
   • “Set read groups information?”: Do not set
   • “Select analysis mode”: 1.Simple Illumina mode

Remove duplicates with MarkDuplicates

MarkDuplicates tool removes duplicate sequences originating from library preparation artifacts and sequencing artifacts. It is important to remove these artefactual sequences to avoid artificial overrepresentation of single molecule.

Hands-on: Remove PCR duplicates

1. MarkDuplicates tool with the following parameters:
   • param-file “Select SAM/BAM dataset or dataset collection”: bam_output (output of Map with BWA-MEM tool)
   • “If true do not write duplicates to the output file instead of writing them with appropriate flags set”: Yes

Generate alignment statistics with Samtools stats

After the duplicate marking step above we can generate statistic about the alignment we have generated.

Hands-on: Generate alignment statistics

1. Samtools stats tool with the following parameters:
   • param-file “BAM file”: outFile (output of MarkDuplicates tool)
   • “Set coverage distribution”: No
   • “Output”: One single summary file
   • “Filter by SAM flags”: Do not filter
   • “Use a reference sequence”: No
   • “Filter by regions”: No

Realign reads with lofreq viterbi

Realign reads tool corrects misalignments around insertions and deletions. This is required in order to accurately detect variants.

Hands-on: Realign reads around indels

1. Realign reads with lofreq tool with the following parameters:
   • param-file “Reads to realign”: outFile (output of MarkDuplicates tool)
   • “Choose the source for the reference genome”: History
     • param-file “Reference”: output (Input dataset)
   • In “Advanced options”:
     • “How to handle base qualities of 2?”: Keep unchanged

Add indel qualities with lofreq Insert indel qualities

This step adds indel qualities into our alignment file. This is necessary in order to call variants using Call variants with lofreq tool

Hands-on: Add indel qualities

1. Insert indel qualities with lofreq tool with the following parameters:
   • param-file “Reads”: realigned (output of Realign reads tool)
   • “Indel calculation approach”: Dindel
     • “Choose the source for the reference genome”: History
       • param-file “Reference”: output (Input dataset)

Call Variants using lofreq Call variants

We are now ready to call variants.

Hands-on: Call variants

1. Call variants with lofreq tool with the following parameters:
   • param-file “Input reads in BAM format”: output (output of Insert indel qualities tool)
   • “Choose the source for the reference genome”: History
     • param-file “Reference”: output (Input dataset)
   • “Call variants across”: Whole reference
   • “Types of variants to call”: SNVs and indels
   • “Variant calling parameters”: Configure settings
     • In “Coverage”:
       • “Minimal coverage”: 50
     • In “Base-calling quality”:
       • “Minimum baseQ”: 30
       • “Minimum baseQ for alternate bases”: 30
     • In “Mapping quality”:
       • “Minimum mapping quality”: 20
   • “Variant filter parameters”: Preset filtering on QUAL score + coverage + strand bias (lofreq call default)

The output of this step is a collection of VCF files that can be visualized in a genome browser.

Annotate variant effects with SnpEff eff:

We will now annotate the variants we called in the previous step with the effect they have on the SARS-CoV-2 genome.

Hands-on: Annotate variant effects

1. SnpEff eff: tool with the following parameters:
   • param-file “Sequence changes (SNPs, MNPs, InDels)”: variants (output of Call variants tool)
   • “Output format”: VCF (only if input is VCF)
   • “Create CSV report, useful for downstream analysis (-csvStats)”: Yes
   • “Annotation options”: ``
   • “Filter output”: ``
   • “Filter out specific Effects”: No

The output of this step is a VCF file with added variant effects.

Create table of variants using SnpSift Extract Fields

We will now select various effects from the VCF and create a tabular file that is easier to understand for humans.

Hands-on: Create table of variants

1. SnpSift Extract Fields tool with the following parameters:
   • param-file “Variant input file in VCF format”: snpeff_output (output of SnpEff eff: tool)
   • “Fields to extract”: CHROM POS REF ALT QUAL DP AF SB DP4 EFF[*].IMPACT EFF[*].FUNCLASS EFF[*].EFFECT EFF[*].GENE EFF[*].CODON
   • “multiple field separator”: ,
   • “empty field text”: .

Summarize data with MultiQC

We will now summarize our analysis with MultiQC, which generates a beautiful report for our data.

Hands-on: Summarize data

1. MultiQC tool with the following parameters:
   • In “Results”:
     • param-repeat “Insert Results”
       • “Which tool was used generate logs?”: fastp
         • param-file “Output of fastp”: report_json (output of fastp tool)
     • param-repeat “Insert Results”
       • “Which tool was used generate logs?”: Samtools
         • In “Samtools output”:
           • param-repeat “Insert Samtools output”
             • “Type of Samtools output?”: stats
               • param-file “Samtools stats output”: output (output of Samtools stats tool)
     • param-repeat “Insert Results”
       • “Which tool was used generate logs?”: Picard
         • In “Picard output”:
           • param-repeat “Insert Picard output”
             • “Type of Picard output?”: Markdups
             • param-file “Picard output”: metrics_file (output of MarkDuplicates tool)
     • param-repeat “Insert Results”
       • “Which tool was used generate logs?”: SnpEff
         • param-file “Output of SnpEff”: csvFile (output of SnpEff eff: tool)

Conclusion

You have seen now how to import public sequencing data into Galaxy, and you have performed the basic steps of variant calling on a batch of RNA-Seq SARS-CoV-2 data.

Galaxy allows you to do a lot more in terms of viral sequence data analysis though. Specifically, the advanced tutorial Mutation calling, viral genome reconstruction and lineage/clade assignment from SARS-CoV-2 sequencing data is highly recommended if you want to learn how to perform a complete and more automated analysis of SARS-CoV-2 sequencing data that does not stop at lists of identified mutations but also produces consensus sequences and lineage assignments for all samples in a batch. This tutorial also shows how to deal with input data other than Illumina paired-end-sequenced RNA-Seq data.

You've Finished the Tutorial

Please also consider filling out the Feedback Form as well!

Key points

• Sequence data in the SRA can be directly imported into Galaxy

• Collections enable efficient/parallel processing of sequence data from whole batches of samples

• Genomic mutations in SARS-CoV-2 viral isolates can be identified through a straightforward pipeline involving sequenced reads preprocessing, mapping to the SARS-CoV-2 reference genome, postprocessing of mapping results, and variant calling

Frequently Asked Questions

Useful literature

Further information, including links to documentation and original publications, regarding the tools, analysis techniques and the interpretation of results described in this tutorial can be found here.

References

1. Lemieux, J. E., K. J. Siddle, B. M. Shaw, C. Loreth, S. F. Schaffner et al., 2021 Phylogenetic analysis of SARS-CoV-2 in Boston highlights the impact of superspreading events. Science 371: 10.1126/science.abe3261

Feedback

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Citing this Tutorial

1. Marius van den Beek, Dave Clements, Daniel Blankenberg, Anton Nekrutenko, From NCBI's Sequence Read Archive (SRA) to Galaxy: SARS-CoV-2 variant analysis (Galaxy Training Materials). https://training.galaxyproject.org/training-material/topics/variant-analysis/tutorials/sars-cov-2/tutorial.html Online; accessed TODAY
2. Hiltemann, Saskia, Rasche, Helena et al., 2023 Galaxy Training: A Powerful Framework for Teaching! PLOS Computational Biology 10.1371/journal.pcbi.1010752
3. Batut et al., 2018 Community-Driven Data Analysis Training for Biology Cell Systems 10.1016/j.cels.2018.05.012

BibTeX

@misc{variant-analysis-sars-cov-2,
author = "Marius van den Beek and Dave Clements and Daniel Blankenberg and Anton Nekrutenko",
	title = "From NCBI's Sequence Read Archive (SRA) to Galaxy: SARS-CoV-2 variant analysis (Galaxy Training Materials)",
	year = "",
	month = "",
	day = "",
	url = "\url{https://training.galaxyproject.org/training-material/topics/variant-analysis/tutorials/sars-cov-2/tutorial.html}",
	note = "[Online; accessed TODAY]"
}
@article{Hiltemann_2023,
	doi = {10.1371/journal.pcbi.1010752},
	url = {https://doi.org/10.1371%2Fjournal.pcbi.1010752},
	year = 2023,
	month = {jan},
	publisher = {Public Library of Science ({PLoS})},
	volume = {19},
	number = {1},
	pages = {e1010752},
	author = {Saskia Hiltemann and Helena Rasche and Simon Gladman and Hans-Rudolf Hotz and Delphine Larivi{\`{e}}re and Daniel Blankenberg and Pratik D. Jagtap and Thomas Wollmann and Anthony Bretaudeau and Nadia Gou{\'{e}} and Timothy J. Griffin and Coline Royaux and Yvan Le Bras and Subina Mehta and Anna Syme and Frederik Coppens and Bert Droesbeke and Nicola Soranzo and Wendi Bacon and Fotis Psomopoulos and Crist{\'{o}}bal Gallardo-Alba and John Davis and Melanie Christine Föll and Matthias Fahrner and Maria A. Doyle and Beatriz Serrano-Solano and Anne Claire Fouilloux and Peter van Heusden and Wolfgang Maier and Dave Clements and Florian Heyl and Björn Grüning and B{\'{e}}r{\'{e}}nice Batut and},
	editor = {Francis Ouellette},
	title = {Galaxy Training: A powerful framework for teaching!},
	journal = {PLoS Comput Biol}
}

                   

Galaxy Administrators: Install the missing tools

You can use Ephemeris's shed-tools install command to install the tools used in this tutorial.

shed-tools install [-g GALAXY] [-a API_KEY] -t <(curl https://training.galaxyproject.org/training-material/api/topics/variant-analysis/tutorials/sars-cov-2/tutorial.json | jq .admin_install_yaml -r)

Alternatively you can copy and paste the following YAML

---
install_tool_dependencies: true
install_repository_dependencies: true
install_resolver_dependencies: true
tools:
- name: text_processing
  owner: bgruening
  revisions: d698c222f354
  tool_panel_section_label: Text Manipulation
  tool_shed_url: https://toolshed.g2.bx.psu.edu/
- name: bwa
  owner: devteam
  revisions: 3fe632431b68
  tool_panel_section_label: Mapping
  tool_shed_url: https://toolshed.g2.bx.psu.edu/
- name: picard
  owner: devteam
  revisions: a1f0b3f4b781
  tool_panel_section_label: Picard
  tool_shed_url: https://toolshed.g2.bx.psu.edu/
- name: samtools_stats
  owner: devteam
  revisions: 145f6d74ff5e
  tool_panel_section_label: SAM/BAM
  tool_shed_url: https://toolshed.g2.bx.psu.edu/
- name: fastp
  owner: iuc
  revisions: dbf9c561ef29
  tool_panel_section_label: FASTA/FASTQ
  tool_shed_url: https://toolshed.g2.bx.psu.edu/
- name: lofreq_call
  owner: iuc
  revisions: 65432c3abf6c
  tool_panel_section_label: Variant Calling
  tool_shed_url: https://toolshed.g2.bx.psu.edu/
- name: lofreq_indelqual
  owner: iuc
  revisions: 426d707dfc47
  tool_panel_section_label: Variant Calling
  tool_shed_url: https://toolshed.g2.bx.psu.edu/
- name: lofreq_viterbi
  owner: iuc
  revisions: aa35ee7f3ab2
  tool_panel_section_label: Variant Calling
  tool_shed_url: https://toolshed.g2.bx.psu.edu/
- name: multiqc
  owner: iuc
  revisions: bf675f34b056
  tool_panel_section_label: Quality Control
  tool_shed_url: https://toolshed.g2.bx.psu.edu/
- name: snpeff_sars_cov_2
  owner: iuc
  revisions: 2a3a00c1fa0a
  tool_panel_section_label: Variant Calling
  tool_shed_url: https://toolshed.g2.bx.psu.edu/
- name: snpsift
  owner: iuc
  revisions: 5fab4f81391d
  tool_panel_section_label: Variant Calling
  tool_shed_url: https://toolshed.g2.bx.psu.edu/
- name: sra_tools
  owner: iuc
  revisions: 494b2ec08162
  tool_panel_section_label: Get Data
  tool_shed_url: https://toolshed.g2.bx.psu.edu/
- name: sra_tools
  owner: iuc
  revisions: 4df8de2d0e48
  tool_panel_section_label: Get Data
  tool_shed_url: https://toolshed.g2.bx.psu.edu/

Feedback

5 stars	4
4 stars	4
2 stars	1
1 stars	1

October 2024

• 4 stars: Liked: The flow follows very nicely and reinforces what we have done on the command line Disliked: AT the snpeff stage there are no instructions on how to get the sars data base. as far as i can tell
• 4 stars: Liked: Most of the tutorial was instructive Disliked: I was lost on the last section, tried different options, got errors, but manage to finished the tutorial

August 2024

• 4 stars: Liked: It was a good walkthrough of how to do variant analysis. I will use it on my class Disliked: The SnpEFF step doesn't say to use the specific covid version of Snpeff. I submitted an earlier response about this, but then found the covid specific too and it worked fine. Maybe just change the tutorial so it points to this. Also some discussion of the multiqc results would be helpful.

June 2023

• 5 stars: Liked: Easy to follow Disliked: Nothing to improve

September 2021

• 4 stars: Liked: how to work on my on data

August 2021

• 1 stars: Liked: terrible Disliked: instructions