Optimized and standardized 192-plex solution for 16S rDNA gene sequencing on Illumina Miseq platform to assess soil biodiversity

The growing need to survey the tremendous microbial diversity in a culture independent manner, has led to the development of molecular methods through sequence profiling of conserved genes such as 16S rDNA. Next-generation sequencing technologies are now used routinely to assess bacterial communities composition in complex environmental samples. Recently, the improvement of the Illumina MiSeq platform to a 2×300 bases paired-end version made it much more attractive for 16S rDNA amplicons sequencing enabling the analysis of a longer region thus facilitating taxonomic assignation. Additionally, the Illumina V3 chemistry enables high-throughput metagenomics analysis at the greatest coverage yet possible with a lower cost per sequence. Nevertheless, known limitations associated with the sequencing of low sequence diversity samples have hampered harnessing its true potential to sequence 16S rDNA gene amplicons. Moreover, validated Illumina 16S rDNA amplicon sequencing protocols do not support more than 96 samples per run, which underutilized the overall capacity of a sequencing run. We therefore worked on the development of a standardized and optimized high-multiplexed metagenomic solution for the exploration of complex environments. We developed an integrated solution combining several technical and bioinformatical strategies that are (i) a custom and standardized amplification protocol for 16S rDNA library preparation to minimize as full as possible technical biases; (ii) a custom 192-index strategy within a single run; (iii) the inclusion of a nucleotide sequence variability at the first sequencing cycles; (iv) an accurate paired-end reads assembly process for full length amplicon analysis and (v) a dedicated pipeline for taxonomic affiliation of 16S rDNA sequences and microbial communities analysis. Our solution validated within soil samples provides high sequencing accuracy and technical reproducibility with no index biases based on taxonomic distribution analysis. The association of technical and bioinformatical improvements yields substantial cost reductions and provides greater target flexibility to assess soil biodiversity as well as functions of microbial communities through carbon, phosphorus or nitrogen cycles in future developments

Reconstructing past vegetation communities using ancient DNA from lake sediments

The field of ancient DNA has received much attention since the mid-1980s, when the first sequences of extinct species were obtained from museum and archaeological specimens. Early analyses focused on organellar DNA (mitochondrial in animals and chloroplast in plants) as these are present in multiple copies in the cells making isolation and analyses easier. Within the last decade, however, with considerable advances in high-throughput DNA sequencing technology and bioinformatics, it has become possible to analyse the more informative nuclear genome of a larger number of ancient samples and from a larger variety of substrates and environments. Here, we present recent progress made to reconstruct ancient vegetation communities from lake sediments and review recent key findings in the field. We synthesize and discuss the sources of plant DNA in sediment, the issues relating to DNA preservation after deposition, the criteria required for authentication and the technical advances recently made in the field for the analyses and the taxonomic identification of plant ancient DNA sequences obtained from these complex substrates. Together, these advances mean that we are on the way to an explosion of new information for the investigation of ancient plant environments