FASTQINS and ANUBIS: two bioinformatic tools to explore facts and artifacts in transposon sequencing and essentiality studies

Miravet-Verde, Samuel; Burgos, Raul; Delgado, Javier; Lluch‐Senar, María; Serrano, Luís

doi:10.1093/nar/gkaa679

Cited by 18 publications

(43 citation statements)

References 50 publications

(84 reference statements)

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“…Table 1), which across the 816 Kb genome leads to a genome coverage of 43.5%, increasing to 64.1% when considering only known non-essential genes (Lluch-Senar et al, 2015). This represented an insertion every ~3 bases, an insertion frequency similar to what has been described in the latest transposition experiments using the same transposase and strain (Miravet-Verde et al, 2020). Figure 1 shows the insertion pattern of the pMTnLox66Cm transposon, along with the second pMTnLox71Tc transposon.…”

Section: Obtaining a High Resolution Library Of Lox Mutantssupporting

confidence: 58%

“…Insertion sites for the transposon were identified using Oligo 19 (Suppl . Table 10), which is bound to the 3' end of the chloramphenicol resistance genes, directly downstream of the inverted repeat (IR) sequence, and identified in the M. pneumoniae M129 genome using FASTQINS (Miravet-Verde et al, 2020). This bioinformatic tool allows to identify the point of insertion of the transposon used in the study and quantify, by read counts, how many times that specific insertion is detected by sequencing.…”

Section: Quantification Of Transposon Densitymentioning

confidence: 99%

“…Table 1). These results were later analysed using ANUBIS essentiality framework (Miravet-Verde et al, 2020) to explore the coverage (i.e. percentage of nucleotide bases found inserted for a considered set of positions), considering the genome of M. pneumoniae (816,394 bp) and for each set of known essential and non-essential genes as described by…”

Section: Quantification Of Transposon Densitymentioning

confidence: 99%

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LoxTnSeq: Random Transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions

Shaw

Miravet-Verde

Piñero

et al. 2020

Preprint

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The Cre-Lox system is a highly versatile and powerful DNA recombinase mechanism, mainly used in genetic engineering to insert or remove desired DNA sequences. It is widely utilised across multiple fields of biology, with applications ranging from plants, to mammals, to microbes. A key feature of this system is its ability to allow recombination between mutant lox sites, traditionally named lox66 and lox71, to create a functionally inactive double mutant lox72 site. However, a large portion of the published literature has incorrectly annotated these mutant lox sites, which in turn can lead to difficulties in replication of methods, design of proper vectors, and confusion over the proper nomenclature. Here, we demonstrate common errors in annotations, the impacts they can have on experimental viability, and a standardised naming convention. We also show an example of how this incorrect annotation can induce toxic effects in bacteria that lack optimal DNA repair systems, exemplified by Mycoplasma pneumoniae.Data SummaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

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Section: Obtaining a High Resolution Library Of Lox Mutantssupporting

confidence: 58%

Section: Quantification Of Transposon Densitymentioning

confidence: 99%

Section: Quantification Of Transposon Densitymentioning

confidence: 99%

See 1 more Smart Citation

LoxTnSeq: Random Transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions

Shaw

Miravet-Verde

Piñero

et al. 2020

Preprint

Self Cite

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“…aureus survival are among the ‘low-counts’ group. While determination of gene essentiality is complex [33–35], we nonetheless conclude that the ‘low-counts’ group contains genes essential for S. aureus survival as 83% of the genes in this group were deemed essential by at least three other reports which incorporated independent studies to deduce essential genes [10, 11, 32].…”

Section: Discussionmentioning

confidence: 93%

Is amplification bias consequential in transposon sequencing (TnSeq) assays? A case study with a Staphylococcus aureus TnSeq library subjected to PCR-based and amplification-free enrichment methods

et al. 2021

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As transposon sequencing (TnSeq) assays have become prolific in the microbiology field, it is of interest to scrutinize their potential drawbacks. TnSeq data consist of millions of nucleotide sequence reads that are generated by PCR amplification of transposon-genomic junctions. Reads mapping to the junctions are enumerated thus providing information on the number of transposon insertion mutations in each individual gene. Here we explore the possibility that PCR amplification of transposon insertions in a TnSeq library skews the results by introducing bias into the detection and/or enumeration of insertions. We compared the detection and frequency of mapped insertions when altering the number of PCR cycles, and when including a nested PCR, in the enrichment step. Additionally, we present nCATRAs - a novel, amplification-free TnSeq method where the insertions are enriched via CRISPR/Cas9-targeted transposon cleavage and subsequent Oxford Nanopore MinION sequencing. nCATRAs achieved 54 and 23% enrichment of the transposons and transposon-genomic junctions, respectively, over background genomic DNA. These PCR-based and PCR-free experiments demonstrate that, overall, PCR amplification does not significantly bias the results of TnSeq insofar as insertions in the majority of genes represented in our library were similarly detected regardless of PCR cycle number and whether or not PCR amplification was employed. However, the detection of a small subset of genes which had been previously described as essential is sensitive to the number of PCR cycles. We conclude that PCR-based enrichment of transposon insertions in a TnSeq assay is reliable, but researchers interested in profiling putative essential genes should carefully weigh the number of amplification cycles employed in their library preparation protocols. In addition, nCATRAs is comparable to traditional PCR-based methods (Kendall’s correlation=0.896–0.897) although the latter remain superior owing to their accessibility and high sequencing depth.

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“…In standard Tn-seq analysis the condition of essentiality is assigned to genes and not to domains, resulting in incorrect classification of many essential genes as non-essential. Rather, essentiality assignment pipeline should be revised to analyze the essentiality of individual protein domains 21 . Indeed, essentiality can be assigned to individual domains of a multidomain protein rather than the entire protein 12,13 .…”

Section: Discussionmentioning

confidence: 99%

Identification of Essential Protein Domains From High-density Transposon Insertion Sequencing

Az¹,

Timmerman²,

Gallardo³

et al. 2021

Preprint

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A first clue to gene function can be obtained by examining whether a gene is required for life in certain standard conditions, that is, whether a gene is essential. In bacteria, essential genes are usually identified by high-density transposon mutagenesis followed by sequencing of insertion sites (Tn-seq). These studies assign the term “essential” to whole genes rather than the protein domain sequences that confer the essential functions. However, genes can code for multiple protein domains that evolve their functions independently. Therefore, when essential genes code for more than one protein domain, only one of them could be essential. In this study, we defined this subset of genes as “essential domain-containing” (EDC) genes. Using a Tn-seq data set built-in Burkholderia cenocepacia K56-2, we developed an in silico pipeline to identify EDC genes and the essential protein domains they encode. We found forty candidate EDC genes and demonstrated growth defect phenotypes using CRISPR interference (CRISPRi). This analysis included two knockdowns of genes encoding the protein domains of unknown function DUF2213 and DUF4148. These essential domains are conserved in more than two hundred bacterial species, including human and plant pathogens. Together, our study suggests that essentiality should be assigned to individual protein domains rather than genes, contributing to a first functional characterization of protein domains of unknown function.

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FASTQINS and ANUBIS: two bioinformatic tools to explore facts and artifacts in transposon sequencing and essentiality studies

Cited by 18 publications

References 50 publications

LoxTnSeq: Random Transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions

LoxTnSeq: Random Transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions

Is amplification bias consequential in transposon sequencing (TnSeq) assays? A case study with a Staphylococcus aureus TnSeq library subjected to PCR-based and amplification-free enrichment methods

Identification of Essential Protein Domains From High-density Transposon Insertion Sequencing

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