Software for RNA-Seq analysis on Windows, including creating sample-specific proteoform databases from genomic data
Spritz can be downloaded here.
Spritz uses snakemake and Docker to install and run commandline tools for Next-Generation Sequencing (NGS) analysis. These tools include an adapted version of SnpEff to annotate sequence variations and create an annotated protein database in XML format. The combinatorics of producing full-length proteoforms from these annotations is written in mzLib's VariantApplication class.
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Install Docker Desktop for Windows.
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Allocate resources to Docker. There are two ways to do this, described in the Spritz wiki:
- The recommended method requires Windows 10 version 2004 and is more robust. Here, we allocate computer resources to Docker like any other program.
- The alternate method is available on all Windows versions but is less robust. Here, we allocate computer resources to Docker using a virtual machine that's packaged with Docker.
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Launch Spritz.
Step 1: Input SRA accessions and/or add FASTQ files.
- SRAs are added with the button indicating single-end or paired-end.
- FASTQ files must end with *_1.fastq if single-end, and paired-end sequences must have the same filename other than ending with *_1.fastq and *_2.fastq.
Step 2: Create and customize your Spritz workflow.
Step 3: Run Spritz!
- Environment:
- Windows 10 recommended
- .NET Core 6.0
- 24 GB RAM recommended
- The installer (Spritz.msi) only works on Windows.
Spritz will also work on the commandline within a Unix system (Linux, Mac, WSL on Windows).
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Add SRR629563 to the SRA list.
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Create the Spritz workflow. Select "release-97" and "homo_sapiens."
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Run Spritz!
Monitor progress in the Information textbox. The final database named final/combined.spritz.snpeff.protein.withmods.xml.gz can be used to search MS/MS with MetaMorpheus to find variant peptides and proteoforms, possibly with modifications. We recommend performing 1) Calibration, 2) Global PTM Discovery (G-PTM-D), and 3) Search tasks to get the best results.
The final database named final/combined.spritz.snpeff.protein.fasta is generated to contain variant protein sequences, and it may be used in other search software, such as Proteome Discoverer, ProSight, and MASH Explorer.
The final database named final/combined.spritz.snpeff.protein.withdecoys.fasta is ready for use in MSFragger. It is generated to contain variant protein sequences with decoy protein sequences appended.
If you use this Spritz, please cite:
Spritz: Cesnik, A. J.; Miller, R. M.; Ibrahim, K.; Lu, L.; Millikin, R. J.; Shortreed, M. R.; Frey, B. L.; Smith, L. M. “Spritz: A Proteogenomic Database Engine.” J. Proteome Res. 2021, 20, 4, 1826–1834. https://pubs.acs.org/doi/abs/10.1021/acs.jproteome.0c00407
This pipeline uses the following tools:
sra-toolkit: Leinonen, R.; et al. International Nucleotide Sequence Database Collaboration. The Sequence Read Archive. Nucleic Acids Res. 2011, 39 (Database issue), D19-21. https://doi.org/10.1093/nar/gkq1019.fastp: Chen, S.; et al. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 2018, 34 (17), i884-i890. https://academic.oup.com/bioinformatics/article/34/17/i884/5093234hisat2: Kim, D.; et al. Graph-Based Genome Alignment and Genotyping with HISAT2 and HISAT-Genotype. Nat. Biotechnol. 2019, 37 (8), 907-915. https://doi.org/10.1038/s41587-019-0201-4."samtools: Li, H.; et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009, 25 (16), 2078-2079. https://academic.oup.com/bioinformatics/article/25/16/2078/204688.GATK: McKenna, A.; et al. The Genome Analysis Toolkit: A MapReduce Framework for Analyzing next-Generation DNA Sequencing Data. Genome Res. 2010, 20 (9), 1297-1303. https://doi.org/10.1101/gr.107524.110.SnpEff: Cingolani, P.; et al. A Program for Annotating and Predicting the Effects of Single Nucleotide Polymorphisms, SnpEff: SNPs in the Genome of Drosophila Melanogaster Strain W1118; Iso-2; Iso-3. Fly (Austin) 2012, 6 (2), 80-92. https://doi.org/10.4161/fly.19695.StringTie2: Kovaka, S.; et al. Transcriptome assembly from long-read RNA-seq alignments with StringTie2. Genome Biol 2019, 20 (278), 1-13. https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1910-1