Lotus 1.64 - quadram-institute-bioscience/gmh-sops GitHub Wiki

Methods

Pre-processing of the sequencing output

The quality profile of the raw reads (in FASTQ format) was assessed using Fastp 0.20.0 (Chen 2018), which was also used to remove reads containing ambiguous bases. The locus-specific primers were removed using SeqFu 0.96 (Telatin 2021).

Identification and classification of Operational Taxonomic Units (OTUs)

OTU picking and their taxonomic classification have been performed using Lotus version 1.64 (Hildebrand 2014), using the UPARSE algorithm (Edgar 2013) for clustering and RDP for taxonomic classification (Cole 2005). The reads were classified using the SILVA database (Quast 2013), for 16S ribosomal sequences, and the UNITE database for the ITS1 sequences (Nilsson 2019).

Numerical ecology analyses

Normalization of the feature table and calculation of alpha-diversity and beta-diversity metrics has been performed according to the "Rhea" protocol, and its R scripts (Lagkouvardos 2017).

References

  • Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018 Sep 1;34(17):i884-i890. doi: 10.1093/bioinformatics/bty560. PMID: 30423086;
  • Telatin A, Fariselli P, Birolo G. SeqFu: A Suite of Utilities for the Robust and Reproducible Manipulation of Sequence Files. Bioengineering (Basel, Switzerland). 2021 May;8(5). DOI: 10.3390/bioengineering8050059. PMID: 34066939
  • Hildebrand F, Tadeo R, Voigt AY, Bork P, Raes J. LotuS: an efficient and user-friendly OTU processing pipeline. Microbiome. 2014 Sep;2(1):30. DOI: 10.1186/2049-2618-2-30. PMID: 27367037
  • Edgar RC. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nature Methods. 2013 Oct;10(10):996-998. DOI: 10.1038/nmeth.2604. PMID: 23955772.
  • Cole JR, Chai B, Farris RJ, et al. The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis. Nucleic Acids Research. 2005 Jan;33(Database issue):D294-6. DOI: 10.1093/nar/gki038. PMID: 15608200; PMCID: PMC539992.
  • Quast C, Pruesse E, Yilmaz P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Research. 2013 Jan;41(Database issue):D590-6. DOI: 10.1093/nar/gks1219. PMID: 23193283; PMCID: PMC3531112.
  • Nilsson RH, Larsson KH, Taylor AFS, et al. The UNITE database for molecular identification of fungi: handling dark taxa and parallel taxonomic classifications. Nucleic Acids Research. 2019 Jan;47(D1):D259-D264. DOI: 10.1093/nar/gky1022. PMID: 30371820; PMCID: PMC6324048.
  • Lagkouvardos I, Fischer S, Kumar N, Clavel T. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons. Peerj. 2017 ;5:e2836. DOI: 10.7717/peerj.2836. PMID: 28097056

If using cutadapt, cite also

  • Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads, EMBnet.journal, 2011. DOI:10.14806/ej.17.1.200