An integrative protocol for one‑step PCR amplicon library construction and accurate demultiplexing of pooled sequencing data

High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures,greatly promoting biological studies involving large amounts of complex samples,especially those involving environmental and pathogen-monitoring ones.Commercial library preparation kits for amplicon sequencing,which generally require multiple steps,including adapter ligation and indexing,are expensive and time-consuming,especially for applications at a large scale.To overcome these limitations,a“one-step PCR approach”has been previously proposed for constructions of amplicon libraries using long fusion primers.However,efficient amplifications of target genes and accurate demultiplexing of pooled sequencing data remain to be addressed.To tackle these,we present an integrative protocol for one-step PCR amplicon library construction(OSPALC).High-quality reads have been generated by this approach to reliably identify species compositions of mock bacterial communities and environmental samples.With this protocol,the amplicon library is constructed through one regular PCR with long primers,and the total cost per DNA/cDNA sample decreases to just 7%of the typical cost via the multi-step PCR approach.Empirically tested primers and optimized PCR conditions to construct OSPALC libraries for 16S rDNA V4 regions are demonstrated as a case study.Tools to design primers targeting at any genomic regions are also presented.In principle,OSPALC can be readily applied to construct amplicon libraries of any target genes using DNA or RNA samples,and will facilitate research in numerous fields.

Detection of integron integrase genes on King George Island, Antarctica

The presence and diversity of class 1 integrase gene （intl） sequences were evaluated by PCR using previously designed primers. Two clone libraries were constructed from DNA in sediment and microbial mat samples collected on Fildes Peninsula, King George Island, Antarctica.The libraries constructed from samples collected at Halfthree Point （HP） and Norma Cove （NC） contained 62 and 36 partial intl sequences, respectively. These sequences clustered into 10 different groups with 〈 95% amino acid identity. Alignment of the deduced amino acid sequences with those from recognized integron-encoded integrases demonstrated the presence of highly conserved motifs characteristic of intl integrases. The HP library contained 42 nucleotide sequences identical to the class 1 intl gene found in a collection of trimethoprim-resistant （Tmpr） Antarctic Enterobacter sp. isolates, previously collected in the same area. These integrons, located on plasmids, had a genetic organization similar to that of pKOX105 from Klebsiella oxytoca. The 20 remaining HP and NC library sequences were similar to integrase sequences previously determined in a metagenomic analysis of environmental samples. We have demonstrated the presence of integron integrase genes in Antarctic sediment samples. About half these genes were very similar to the class 1 integrons found in human- associated microbiota, suggesting that they originated from human-dominated ecosystems. The remaining integrase genes were probably associated with endemic bacteria.

Evaluation of Molecular Techniques in Characterization of Deep Terrestrial Biosphere

A suite of molecular methods targeting 16S rRNA genes (i.e., DGGE, clone and high-throughput [HTP] amplicon library sequencing) was used to profile the microbial communities in deep Fennoscandian crystalline bedrock fracture fluids. Variation among bacterial 16S rRNA genes was examined with two commonly used primer pairs: P1/P2 and U968f/U1401r. DGGE using U968f/ U1401r mostly detected β-, γ-proteobacteria and Firmicutes, while P1/P2 primers additionally detected other proteobacterial clades and candidate divisions. However, in combination with clone libraries the U968f/U1401r primers detected a higher bacterial diversity than DGGE alone. HTP amplicon sequencing with P1/P2 revealed an abundance of the DGGE bacterial groups as well as many other bacterial taxa likely representing minor components of these communities. Archaeal diversity was investigated via DGGE or HTP amplicon sequencingusing primers A344F/ 519RP. The majority of archaea detected with HTP amplicon sequencing belonged to uncultured Thermoplasmatales and Pendant 33/DHVE3, 4, 6 groups. DGGE of the same samples detected mostly SAGMEG and Methanosarcinales archaea, but almost none of those were revealed by HTP amplicon sequencing. Overall, our results show that the inferred diversity and composition of microbial communities in deep fracture fluids is highly dependent on analytical technique and that the method should be carefully selected with this in mind.