ddPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samples.

Background: One limiting factor of short amplicon 16S rRNA gene sequencing approaches is the use of low DNA amounts in the amplicon generation step. Especially for low-biomass samples, insufficient or even commonly undetectable DNA amounts can limit or prohibit further analysis in standard protocols.

Results: Using a newly established protocol, very low DNA input amounts were found sufficient for reliable detection of bacteria using 16S rRNA gene sequencing compared to standard protocols.

Full-Length SSU rRNA Gene Sequencing Allows Species-Level Detection of Bacteria, Archaea, and Yeasts Present in Milk.

Full-length SSU rRNA gene sequencing allows species-level identification of the microorganisms present in milk samples. Here, we used bulk-tank raw milk samples of two German dairies and detected, using this method, a great diversity of bacteria, archaea, and yeasts within the samples. Moreover, the species-level classification was improved in comparison to short amplicon sequencing.

Primer, Pipelines, Parameters: Issues in 16S rRNA Gene Sequencing.

Short-amplicon 16S rRNA gene sequencing is currently the method of choice for studies investigating microbiomes. However, comparative studies on differences in procedures are scarce. We sequenced human stool samples and mock communities with increasing complexity using a variety of commonly used protocols.

The Novel Anaerobiosis-Responsive Overlapping Gene Is Overlapping Antisense to the Annotated Gene ECs2385 of O157:H7 Sakai.

Current notion presumes that only one protein is encoded at a given bacterial genetic locus. However, transcription and translation of an overlapping open reading frame (ORF) of 186 bp length were discovered by RNAseq and RIBOseq experiments. This ORF is almost completely embedded in the annotated L,D-transpeptidase gene ECs2385 of O157:H7 Sakai in the antisense reading frame -3.

A novel short L-arginine responsive protein-coding gene (laoB) antiparallel overlapping to a CadC-like transcriptional regulator in Escherichia coli O157:H7 Sakai originated by overprinting.

Background: Due to the DNA triplet code, it is possible that the sequences of two or more protein-coding genes overlap to a large degree. However, such non-trivial overlaps are usually excluded by genome annotation pipelines and, thus, only a few overlapping gene pairs have been described in bacteria. In contrast, transcriptome and translatome sequencing reveals many signals originated from the antisense strand of annotated genes, of which we analyzed an example gene pair in more detail.