M20 Genomics
  • Q Is there any restriction on the shape and diameter of bacteria?
    A

    No restriction applies. The diameter of bacteria typically falls between 0.5-10μm, meeting the detection criteria.

  • Q What is the definition of a single bacterium, and does it encompass different strains of the same bacterial species when mixed?
    A

    In this context, a single bacterium refers to an individual bacterium at the species level.

  • Q Does the preparation of single bacterial suspensions from fecal samples require enrichment culturing?
    A

    Pre-treatment of fecal samples: Adding PBST (containing RNase inhibitor), vortexing to mix, filter to remove impurities, then perform high-speed centrifugation to collect the microbial precipitate for subsequent fixation. Enrichment culturing is not required.

  • Q What is the recommended number of bacteria and sequencing depth?
    A

    For single bacterial cells: 2000-3000 cells, sequencing depth 30-60G or 100-200M reads;
    For mixed bacterial cells: 5000-10000 cells, sequencing depth 150-200G or 500M-650M reads.

  • Q How to solve the problem of high rRNA content?
    A

    A higher proportion of rRNA can lead to data loss. Since bacteria have lower nucleic acid content, increasing the sequencing depth can ensure sufficient genes for subsequent analysis without significantly impacting the final clustering and gene expression analysis.

  • Q What advantages does single bacterial transcriptome have compared to bulk RNA-seq of bacteria?
    A

    It can disclose the heterogeneity of bacterial expression levels and reveal differences masked by averaged values.

  • Q Can the transcription-level differences between wild-type and mutated strains be detected after point mutation modification?
    A

    As long as there are differences in expression, they can be detected.

  • Q Can VITA prokaryotic single-cell transcriptome analysis reflect bacterial abundance? How do results compare with those obtained from metatranscriptome sequencing?
    A

    It can reflect bacterial abundance, with minor differences compared to metatranscriptome. The dominant species are similar .

  • Q Is a reference genome required for functional analysis of single bacterial transcriptomes?
    A

    Yes, it is required.

  • Q Can VITA prokaryotic single-cell transcriptome product study bacterial interactions?
    A

    Yes, testing for multiple bacterial interactions has already been achieved.

  • Q After identification of bacterial subgroups, is functional validation still necessary?
    A

    Functional validation can be performed based on research objectives, such as constructing fluorescent reporter strains or flow sorting.

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