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Despite the advent of third-generation sequencing technologies, modern bacterial ecology studies still use Illumina to sequence small (~400 bp) hypervariable regions of the 16S rRNA SSU for phylogenetic classification. By sequencing a larger region of the rRNA gene operons, the limitations and biases of sequencing small portions can be removed, allowing for more accurate classification with deeper taxonomic resolution. With Nanopore sequencing now providing raw simplex reads with quality scores above Q20 using the kit 12 chemistry, the ease, cost, and portability of Nanopore play a leading role in performing differential bacterial abundance analysis. Sequencing the near-entire rrn operon of bacteria and archaea enables the use of the universally conserved operon holding evolutionary polymorphisms for taxonomic resolution. Here, a reproducible and validated pipeline was developed, RRN-operon Enabled Species-level Classification Using EMU (RESCUE), to facilitate the sequencing of bacterial rrn operons and to support import into phyloseq. Benchmarking RESCUE showed that fully processed reads are now parallel or exceed the quality of Sanger, with median quality scores of approximately Q20+, using the R10.4 and Guppy SUP basecalling. The pipeline was validated through two complex mock samples, the use of multiple sample types, with actual Illumina data, and across four databases. RESCUE sequencing is shown to drastically improve classification to the species level for most taxa and resolves erroneous taxa caused by using short reads such as Illumina. [ABSTRACT FROM AUTHOR]

Despite the advent of third-generation sequencing technologies, modern bacterial ecology studies still use Illumina to sequence small (~400 bp) hypervariable regions of the 16S rRNA SSU for phylogenetic classification. By sequencing a larger region of the rRNA gene operons, the limitations and biases of sequencing small portions can be removed, allowing for more accurate classification with deeper taxonomic resolution. With Nanopore sequencing now providing raw simplex reads with quality scores above Q20 using the kit 12 chemistry, the ease, cost, and portability of Nanopore play a leading role in performing differential bacterial abundance analysis. Sequencing the near-entire rrn operon of bacteria and archaea enables the use of the universally conserved operon holding evolutionary polymorphisms for taxonomic resolution. Here, a reproducible and validated pipeline was developed, RRN-operon Enabled Species-level Classification Using EMU (RESCUE), to facilitate the sequencing of bacterial rrn operons and to support import into phyloseq. Benchmarking RESCUE showed that fully processed reads are now parallel or exceed the quality of Sanger, with median quality scores of approximately Q20+, using the R10.4 and Guppy SUP basecalling. The pipeline was validated through two complex mock samples, the use of multiple sample types, with actual Illumina data, and across four databases. RESCUE sequencing is shown to drastically improve classification to the species level for most taxa and resolves erroneous taxa caused by using short reads such as Illumina.

Aims To perform a systematic evaluation of the applicability, validity and reliability of the long PCR- RFLP of 16 S- ITS-23 S r RNA genes for bacterial genotyping using both sequences retrieved from public genome databases and the experimental data obtained on bacterial cultures. Methods and Results 3301 Full-length sequences of 16 S- ITS-23 S r RNA genes were retrieved from 885 published bacterial genomes. Copy numbers of the whole set of 16 S- ITS-23 S r RNA genes per genome ranged from 1 ( n = 161) to 14 ( n = 4) with an average of 3·71. Their length varied greatly, from 4319 to 6568 bp with an average of 4952 bp. Computer-simulated RFLP analyses of the 16 S- ITS-23 S fragments flanked by the conserved primers 27 F and 2241 R suggested Msp I, Rsa I, Hha I and Taq I as the most appropriate enzymes for long PCR- RFLP analysis of the 16 S- ITS-23 S sequence. Msp I was used to screen over 900 bacterial cultures isolated from the Huguangyan Maar Lake in southern China. An experimental sequencing of 16 S r RNA genes of the isolates possessing a unique RFLP band pattern proved the broad applicability and high resolution of this approach. Conclusions These results indicate that long PCR- RFLP of 16 S- ITS-23 S r RNA genes is a potentially universal and reliable bacterial genotyping tool with a high resolution. Significance and Impact of the Study The methodology of long PCR- RFLP of 16 S- ITS-23 S r RNA genes will facilitate the exploration and tracing of cultivable microbial diversity in natural environments. [ABSTRACT FROM AUTHOR]