Genomic and Phenotypic Analysis of COVID-19-Associated Pulmonary Aspergillosis Isolates of Aspergillus fumigatus

  1. Jacob L. Steenwyk
  2. Matthew E. Mead
  3. Patrícia Alves de Castro
  4. Clara Valero
  5. André Damasio
  6. Renato A. C. dos Santos
  7. Abigail L. Labella
  8. Yuanning Li
  9. Sonja L. Knowles
  10. Huzefa A. Raja
  11. Nicholas H. Oberlies
  12. Xiaofan Zhou
  13. Oliver A. Cornely
  14. Frieder Fuchs
  15. Philipp Koehler
  16. Gustavo H. Goldman
  17. Antonis Rokas

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Abstract

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), has already killed millions of people. COVID-19 patient outcome can be further complicated by secondary infections, such as COVID-19-associated pulmonary aspergillosis (CAPA).

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  1. Version published to 10.1128/spectrum.00010-21
  2. ScreenIT

    SciScore for 10.1101/2020.11.06.371971: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    For each sample, 1-1.5μg genomic DNA was randomly fragmented by Covaris and fragments with average size of 200-400bp were selected by Agencourt AMPure XP-Medium kit.
    Covaris
    suggested: None
    Genome assembly and annotations: Short-read sequencing data of each sample were assembled using MaSuRCA, v3.4.1 (Zimin et al., 2013).
    MaSuRCA
    suggested: (MaSuRCA, RRID:SCR_010691)
    The MAKER annotation process occurs in an iterative manner as described previously (Shen et al., 2018).
    MAKER
    suggested: (MAKER, RRID:SCR_005309)
    In brief, for each genome, repeats were first soft-masked using RepeatMasker v4.1.0 (http://www.repeatmasker.org) with the library Repbase library release-20181026 and the “-species” parameter set to “Aspergillus fumigatus”.
    RepeatMasker
    suggested: (RepeatMasker, RRID:SCR_012954)
    Repbase
    suggested: None
    On the other hand, an initial MAKER analysis was carried out where gene annotations were generated directly from homology evidence, and the resulting gene models were used to train both AUGUSTUS and SNAP.
    AUGUSTUS
    suggested: (Augustus, RRID:SCR_008417)
    Resulting gene models supported by homology evidence were used to re-train AUGUSTUS and SNAP.
    SNAP
    suggested: (SNAP, RRID:SCR_007936)
    The completeness of de novo genome assemblies and ab initio gene predictions was assessed using BUSCO, v4.1.2 (Waterhouse et al., 2018) using 4,191 pre-selected ‘nearly’ universally single-copy orthologous genes from the Eurotiales database (eurotials_odb10.2019-11-20) in OrthoDB, v10.1 (Waterhouse et al.,
    BUSCO
    suggested: (BUSCO, RRID:SCR_015008)
    OrthoDB
    suggested: (OrthoDB, RRID:SCR_011980)
    To do so, reads were first quality-trimmed and mapped to the genome of A. fumigatus Af293 (RefSeq assembly accession: GCF_000002655.1) following a previously established protocol (Steenwyk and Rokas, 2017).
    RefSeq
    suggested: (RefSeq, RRID:SCR_003496)
    Specifically, reads were first quality-trimmed with Trimmomatic, v0.36 (Bolger et al., 2014), using the parameters leading:10, trailing:10, slidingwindow:4:20, minlen:50.
    Trimmomatic
    suggested: (Trimmomatic, RRID:SCR_011848)
    The resulting quality-trimmed reads were mapped to the A. fumigatus Af293 genome using the Burrows-Wheeler Aligner (BWA),
    BWA
    suggested: (BWA, RRID:SCR_010910)
    Thereafter, mapped reads were converted to a sorted bam and mpileup format for polymorphism identification using SAMtools, v.1.3.1 (Li et al., 2009).
    SAMtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    To identify SNPs and indels, mpileup files were used as input into VarScan, v2.3.9 (Koboldt et al., 2012), with the mpileup2snp and mpileup2indel functions, respectively.
    VarScan
    suggested: (VARSCAN, RRID:SCR_006849)
    The resulting Variant Call Format files were used as input to snpEff, v.4.3t (Cingolani et al., 2012), which predicted their functional impacts on gene function as high, moderate, or low.
    snpEff
    suggested: (SnpEff, RRID:SCR_005191)
    The coefficientOfVariation parameter was set to 0.062 and window size was automatically determined by Control-FREEC.
    Control-FREEC
    suggested: (Control-FREEC, RRID:SCR_010822)
    In the first analysis, the nucleotide sequence of the alpha subunit of translation elongation factor EF-1, tef1 (NCBI Accession: XM_745295.2), from the genome of Aspergillus fumigatus Af293 was used to extract other fungal tef1 sequences from NCBI’s fungal nucleotide reference sequence database (downloaded July 2020) using the blastn function from NCBI’s BLAST+, v2.3.0 (Camacho et al., 2009).
    BLAST+
    suggested: (Japan Bioinformatics, RRID:SCR_012250)
    Tef1 sequences were extracted from the CAPA isolates by identifying their best BLAST hit.
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    To do so, we first identified orthologous groups of genes across all 50 Aspergillus using OrthoFinder, 2.3.8 (Emms and Kelly, 2019).
    OrthoFinder
    suggested: (OrthoFinder, RRID:SCR_017118)
    OrthoFinder takes as input the proteome sequence files from multiple genomes and conducts all-vs-all sequence similarity searches using DIAMOND, v0.9.24.125 (Buchfink et al., 2015).
    DIAMOND
    suggested: (DIAMOND, RRID:SCR_009457)
    To do so, the protein sequences from 4,525 single-copy orthologous groups of genes were aligned using MAFFT, v7.402 (Katoh and Standley, 2013), with the following parameters: --bl 62 --op 1.0 --maxiterate 1000 --retree 1 --genafpair.
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    ), BLAST-based searches using an expectation value threshold of 1e-10 were used to identify BGCs implicated in modulating host biology using NCBI’s BLAST+, v2.3.0 (Camacho et al., 2009).
    NCBI’s BLAST+
    suggested: None
    Data Availability: Newly sequenced genomes assemblies, annotations, and raw short reads have been deposited to NCBI’s GenBank database under BioProject accession PRJNA673120.
    BioProject
    suggested: (NCBI BioProject, RRID:SCR_004801)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT01731353RecruitingFungiscope - A Global Emerging Fungal Infection Registry

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

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  3. Version published to 10.1101/2020.11.06.371971v1 on bioRxiv