A Two-Step PCR Protocol Enabling Flexible Primer Choice and High Sequencing Yield for Illumina MiSeq Meta-Barcoding

High-throughput amplicon sequencing that primarily targets the 16S ribosomal DNA (rDNA) (for bacteria and archaea) and the Internal Transcribed Spacer rDNA (for fungi) have facilitated microbial community discovery across diverse environments. A three-step PCR that utilizes flexible primer choices to construct the library for Illumina amplicon sequencing has been applied to several studies in forest and agricultural systems. The three-step PCR protocol, while producing high-quality reads, often yields a large number (up to 46%) of reads that are unable to be assigned to a specific sample according to its barcode. Here, we improve this technique through an optimized two-step PCR protocol. We tested and compared the improved two-step PCR meta-barcoding protocol against the three-step PCR protocol using four different primer pairs (fungal ITS: ITS1F-ITS2 and ITS1F-ITS4, and bacterial 16S: 515F-806R and 341F-806R). We demonstrate that the sequence quantity and recovery rate were significantly improved with the two-step PCR approach (fourfold more read counts per sample; determined reads ≈90% per run) while retaining high read quality (Q30 > 80%). Given that synthetic barcodes are incorporated independently from any specific primers, this two-step PCR protocol can be broadly adapted to different genomic regions and organisms of scientific interest.

Endophytic bacterial communities and spatiotemporal variations in cotton roots in Xinjiang, China

Endophytic bacteria may be important for plant health and other ecologically relevant functions of cotton. However, the endophytic bacterial community structure and diversity in cotton is still poorly characterized. We investigated the community structure of endophytic bacteria in cotton roots growing in Xinjiang, China, using the Illumina amplicon sequencing. A total of 60.84 M effective sequences of 16S rRNA gene V3 region were obtained from cotton samples. These sequences revealed huge amount of operational taxonomic units (OTUs) in cotton, that is, 81-338 OTUs in a cotton sample, at 3% cutoff level and sequencing depth of 50000 sequences. We identified 23 classes from the resulting 2,723,384 sequences. Gammaproteobacteria were the dominant class in all cottons, followed by Alphaproteobacteria, Actinobacteria and Bacilli. A marked difference in the diversity of endophytic bacteria in cotton for different growth periods was evident. The greatest number of OTUs was detected during seedling (654 OTUs) and budding (381 OTUs). Endophytic bacteria diversity was reduced during flowering (350 OTUs) and boll-opening (351 OTUs). 217 OTUs were common to all four periods. There were more tags of Pantoea in Shihezi than other locations. While there were more tags of Erwinia in Hami than other locations. The dynamics of endophytic bacteria communities were influenced by plant growth stage. These results show the complexity of the bacterial populations present in inner tissues of cotton.

Planktonic and Benthic Bacterial Communities of the Largest Central European Shallow Lake, Lake Balaton and Its Main Inflow Zala River

Lake Balaton is the largest European shallow lake, which underwent cultural eutrophication in the ‘70–80s. Therefore, strict pollution control measures were introduced and the water quality has become meso-eutrophic since the millennium. Due to the touristic significance and change in trophic levels of the lake, numerous ecological studies were carried out, but none of them was focused on both benthic and planktonic microbial communities at the same time. In our study, an attempt was made to reveal the spatial bacterial heterogeneity of the Lake Balaton and Zala River by 16S rDNA terminal restriction fragment length polymorphism fingerprinting and Illumina amplicon sequencing methods in the summer of 2017. According to the molecular biology results, mostly well-known freshwater microorganisms, adapted to nutrient-poor conditions were found in the pelagic water column. The LD12 subclade member Fonsibacter ubiquis, the cyanobacterial Synechococcus sp. and unknown Verrucomicrobia species were abundant in the less nutrient-dense basins, while the hgcI clade members showed various distribution. In the estuary and in the nutrient-dense western part of the lake, some eutrophic conditions preferring cyanobacteria (filamentous Anabaena and Aphanizomenon species) were also detectable. The benthic microbial community showed higher diversity, according to the observed appearance of microorganisms adapted to the deeper, less aerated layers (e.g. members of Desulfobacteraceae, Nitrosomonadaceae).

Rapid and cost-effective generation of single specimen multilocus barcoding data from whole arthropod communities by multiple levels of multiplexing

In light of the current biodiversity crisis, molecular barcoding has developed into an irreplaceable tool. Barcoding has been considerably simplified by developments in high throughput sequencing technology, but still can be prohibitively expensive and laborious when community samples of thousands of specimens need to be processed. Here, we outline an Illumina amplicon sequencing approach to generate multilocus data from large collections of arthropods. We reduce cost and effort up to 50-fold, by combining multiplex PCRs and DNA extractions from pools of presorted and morphotyped specimens and using two levels of sample indexing. We test our protocol by generating a comprehensive, community wide dataset of barcode sequences for several thousand Hawaiian arthropods from 14 orders, which were collected across the archipelago using various trapping methods. We explore patterns of diversity across the Archipelago and compare the utility of different arthropod trapping methods for biodiversity explorations on Hawaii, highlighting undergrowth beating as highly efficient method. Moreover, we show the effects of barcode marker, taxonomy and relative biomass of the targeted specimens and sequencing coverage on taxon recovery. Our protocol enables rapid and inexpensive explorations of diversity patterns and the generation of multilocus barcode reference libraries across whole ecosystems.

Comparison of Six Commercial Meat Starter Cultures for the Fermentation of Yellow Mealworm (Tenebrio molitor) Paste

In this study, six commercial meat starters, each consisting of a pure strain of a lactic acid-fermenting bacterium (including Lactococcus lactis, Lactobacillus curvatus, L. farciminis, L. plantarum, L. sakei, and Pediococcus acidilactici), were tested for their ability to ferment a paste produced from the yellow mealworm (Tenebrio molitor). During fermentation, microbial counts, pH, and the bacterial community composition were determined. In addition, UPLC-MS was applied to monitor the consumption of glucose and the production of glutamic (Glu) and aspartic (Asp) acid. All tested starters were able to ferment the mealworm paste, judged by a pH reduction from 6.68 to 4.60–4.95 within 72 h. Illumina amplicon sequencing showed that all starters were able to colonize the substrate efficiently. Moreover, the introduction of the starter cultures led to the disappearance of Bacillus and Clostridium species, which were the dominant microorganisms in un-inoculated samples. Of the six cultures tested, Lactobacillus farciminis was most promising as its application resulted in the largest increase (±25 mg/100 g of paste) in the content of free glutamic and aspartic acid. These amino acids are responsible for the appreciated umami flavour in fermented food products and might stimulate the acceptance of insects and their consumption.