A method to generate multilocus barcodes of pinned insect specimens using MiSeq

For molecular insect identification, amplicon sequencing methods are recommended because they offer a cost‐effective approach for targeting small sets of informative genes from multiple samples. In this context, high‐throughput multilocus amplicon sequencing has been achieved using the MiSeq Illumina sequencing platform. However, this approach generates short gene fragments of <500 bp, which then have to be overlapped using bioinformatics to achieve longer sequence lengths. This increases the risk of generating chimeric sequences or leads to the formation of incomplete loci. Here, we propose a modified nested amplicon sequencing method for targeting multiple loci from pinned insect specimens using the MiSeq Illumina platform. The modification exists in using a three‐step nested PCR approach targeting near full‐length loci in the initial PCR and subsequently amplifying short fragments of between 300 and 350 bp for high‐throughput sequencing using Illumina chemistry. Using this method, we generated 407 sequences of three loci from 86% of all the specimens sequenced. Out of 103 pinned bee specimens of replicated species, 71% passed the 95% sequence similarity threshold between species replicates. This method worked best for pinned specimens aged between 0 and 5 years, with a limit of 10 years for pinned and 14 years for ethanol‐preserved specimens. Hence, our method overcomes some of the challenges of amplicon sequencing using short read next generation sequencing and improves the possibility of creating high‐quality multilocus barcodes from insect collections.     

New barcoded primers for efficient retrieval of cercozoan sequences in high‐throughput environmental diversity surveys,with emphasis on worldwide biological soil crusts

We describe the performance of a new metabarcoding approach to investigate the environmental diversity of a prominent group of widespread unicellular organisms, the Cercozoa. Cercozoa is an immensely large group of protists, and although it may dominate in soil and aquatic ecosystems, its environmental diversity remains undersampled. We designed PCR primers targeting the hypervariable region V4 of the small subunit ribosomal RNA (SSU or 18S) gene, which is the recommended barcode marker for Cercozoa. The length of the amplified fragment (c. 350 bp) is suitable for Illumina MiSeq, the most cost‐effective platform for molecular environmental surveys. We provide barcoded primers, an economical alternative to multiple libraries for multiplex sequencing of over a hundred samples. In silico, our primers matched 68% of the cercozoan sequences of the reference database and performed better than previously proposed new‐generation sequencing primers. In mountain grassland soils and in biological soil crusts from a variety of climatic regions, we were able to detect cercozoan sequences encompassing nearly the whole range of the phylum. We obtained 901 operational taxonomic units (OTUs) at 97% similarity threshold from 26 samples, with c. 50,000 sequences per site, and only 8% of noncercozoan sequences. We could report a further increase in the diversity of Cercozoa, as only 43% of the OTUs were 97%–100% similar to any known sequence. Our study thus provides an advanced tool for cercozoan metabarcoding and to investigate their diversity and distribution in the environment.     

Modular tagging of amplicons using a single PCR for high‐throughput sequencing

High‐throughput sequencing (HTS) of PCR amplicons is becoming the method of choice to sequence one or several targeted loci for phylogenetic and DNA barcoding studies. Although the development of HTS has allowed rapid generation of massive amounts of DNA sequence data, preparing amplicons for HTS remains a rate‐limiting step. For example, HTS platforms require platform‐specific adapter sequences to be present at the 5′ and 3′ end of the DNA fragment to be sequenced. In addition, short multiplex identifier (MID) tags are typically added to allow multiple samples to be pooled in a single HTS run. Existing methods to incorporate HTS adapters and MID tags into PCR amplicons are either inefficient, requiring multiple enzymatic reactions and clean‐up steps, or costly when applied to multiple samples or loci (fusion primers). We describe a method to amplify a target locus and add HTS adapters and MID tags via a linker sequence using a single PCR. We demonstrate our approach by generating reference sequence data for two mitochondrial loci (COI and 16S) for a diverse suite of insect taxa. Our approach provides a flexible, cost‐effective and efficient method to prepare amplicons for HTS.     

Validation of loop‐mediated isothermal amplification for fast and portable sex determination across the phylogeny of birds

PCR is a universal tool for the multiplication of specific DNA sequences. For example, PCR‐based sex determination is widely used, and a diversity of primer sets is available. However, this protocol requires thermal cycling and electrophoresis, so results are typically obtained in laboratories and several days after sampling. Loop‐mediated isothermal amplification (LAMP) is an alternative to PCR that can take molecular ecology outside the laboratory. Although its application has been successfully probed for sex determination in three species of a single avian Family (raptors, Accipitridae), its generality remains untested and suitable primers across taxa are lacking. We designed and tested the first LAMP‐based primer set for sex determination across the modern birds (NEO‐W) based on a fragment of the gene chromo‐helicase‐DNA‐binding protein located on the female‐specific W chromosome. As nucleotide identity is expected to increase among more related taxa, taxonomically targeted primers were also developed for the Order Falconiformes and Families Psittacidae, Ciconiidae, Estrildidae and Icteridae as examples. NEO‐W successfully determined sex in a subset of 21 species within 17 Families and 10 Orders and is therefore a candidate primer for all modern birds. Primer sets designed specifically for the selected taxa correctly assigned sex to the evaluated species. A short troubleshooting guide for new LAMP users is provided to identify false negatives and optimize LAMP reactions. This study represents the crucial next step towards the use of LAMP for molecular sex determination in birds and other applications in molecular ecology.     

One‐step Multiplex RT‐PCR for Simultaneous Detection of Four Viruses in Tobacco

A one‐step multiplex RT‐PCR method has been developed for the simultaneous detection of four viruses frequently occurring in tobacco (Cucumber mosaic virus, Tobacco mosaic virus, Tobacco etch virus and Potato virus Y). Four sets of specific primers were designed to work with the same reaction reagents and cycling conditions, resulting in four distinguishable amplicons representative of the four viruses independently. This one‐step multiplex RT‐PCR is consistently specific using different combinations of virus RNA as templates, and no non‐specific band was observed. It has high sensitivity compared to single RT‐PCR. Moreover, field samples in China can be tested by this method for virus detection. Our results show that one‐step multiplex RT‐PCR is a high‐throughput, specific, sensitive method for tobacco virus detection.     

Tag jumps illuminated – reducing sequence‐to‐sample misidentifications in metabarcoding studies

Metabarcoding of environmental samples on second‐generation sequencing platforms has rapidly become a valuable tool for ecological studies. A fundamental assumption of this approach is the reliance on being able to track tagged amplicons back to the samples from which they originated. In this study, we address the problem of sequences in metabarcoding sequencing outputs with false combinations of used tags (tag jumps). Unless these sequences can be identified and excluded from downstream analyses, tag jumps creating sequences with false, but already used tag combinations, can cause incorrect assignment of sequences to samples and artificially inflate diversity. In this study, we document and investigate tag jumping in metabarcoding studies on Illumina sequencing platforms by amplifying mixed‐template extracts obtained from bat droppings and leech gut contents with tagged generic arthropod and mammal primers, respectively. We found that an average of 2.6% and 2.1% of sequences had tag combinations, which could be explained by tag jumping in the leech and bat diet study, respectively. We suggest that tag jumping can happen during blunt‐ending of pools of tagged amplicons during library build and as a consequence of chimera formation during bulk amplification of tagged amplicons during library index PCR. We argue that tag jumping and contamination between libraries represents a considerable challenge for Illumina‐based metabarcoding studies, and suggest measures to avoid false assignment of tag jumping‐derived sequences to samples.     

Use of allele-specific sequencing primers is an efficient alternative to PCR subcloning of low-copy nuclear genes

Direct Sanger sequencing of polymerase chain reaction (PCR)-amplified nuclear genes leads to polymorphic sequences when allelic variation is present. To overcome this problem, most researchers subclone the PCR products to separate alleles. An alternative is to directly sequence the separate alleles using allele-specific primers. We tested two methods to enhance the specificity of allele-specific primers for use in direct sequencing: using short primers and amplification refractory mutation system (ARMS) technique. By shortening the allele-specific primer to 15-13 nucleotides, the single mismatch in the ultimate base of the primer is enough to hinder the amplification of the nontarget allele in direct sequencing and recover only the targeted allele at high accuracy. The deliberate addition of a second mismatch, as implemented in the ARMS technique, was less successful and seems better suited for allele-specific amplification in regular PCR rather than in direct sequencing.     

Design of character‐based DNA barcode motif for species identification: A computational approach and its validation in fishes

The DNA barcodes are generally interpreted using distance‐based and character‐based methods. The former uses clustering of comparable groups, based on the relative genetic distance, while the latter is based on the presence or absence of discrete nucleotide substitutions. The distance‐based approach has a limitation in defining a universal species boundary across the taxa as the rate of mtDNA evolution is not constant throughout the taxa. However, character‐based approach more accurately defines this using a unique set of nucleotide characters. The character‐based analysis of full‐length barcode has some inherent limitations, like sequencing of the full‐length barcode, use of a sparse‐data matrix and lack of a uniform diagnostic position for each group. A short continuous stretch of a fragment can be used to resolve the limitations. Here, we observe that a 154‐bp fragment, from the transversion‐rich domain of 1367 COI barcode sequences can successfully delimit species in the three most diverse orders of freshwater fishes. This fragment is used to design species‐specific barcode motifs for 109 species by the character‐based method, which successfully identifies the correct species using a pattern‐matching program. The motifs also correctly identify geographically isolated population of the Cypriniformes species. Further, this region is validated as a species‐specific mini‐barcode for freshwater fishes by successful PCR amplification and sequencing of the motif (154 bp) using the designed primers. We anticipate that use of such motifs will enhance the diagnostic power of DNA barcode, and the mini‐barcode approach will greatly benefit the field‐based system of rapid species identification.     

Reconstructing the plant mitochondrial genome for marker discovery: a case study using Pinus

Whole‐genome‐shotgun (WGS) sequencing of total genomic DNA was used to recover ~1 Mbp of novel mitochondrial (mtDNA) sequence from Pinus sylvestris (L.) and three members of the closely related Pinus mugo species complex. DNA was extracted from megagametophyte tissue from six mother trees from locations across Europe, and 100‐bp paired‐end sequencing was performed on the Illumina HiSeq platform. Candidate mtDNA sequences were identified by their size and coverage characteristics, and by comparison with published plant mitochondrial genomes. Novel variants were identified, and primers targeting these loci were trialled on a set of 28 individuals from across Europe. In total, 31 SNP loci were successfully resequenced, characterizing 15 unique haplotypes. This approach offers a cost‐effective means of developing marker resources for mitochondrial genomes in other plant species where reference sequences are unavailable.     

Noncoding mitochondrial loci for corals

We developed five degenerate primer pairs for the amplification and sequencing of two noncoding regions found in the mitochondrial genome of corals. These primers amplify products ranging from 380 to 950 bp, and work in a wide variety of scleractinian taxa from both the Pacific and Caribbean. Based on our initial analysis of ～300 sequences from 13 scleractinian taxa, both these noncoding regions appear to have equivalent levels of variability to the most variable of previously published coral mitochondrial loci, but work in a wider variety of taxa. We believe these primers will be of use to coral biologists studying questions above the level of species; as with other mithochondrial DNA markers in corals, these loci will likely provide little resolution for within‐species studies.     

SPInDel: a multifunctional workbench for species identification using insertion/deletion variants

The majority of the available methods for the molecular identification of species use pairwise sequence divergences between the query and reference sequences (DNA barcoding). The presence of multiple insertions and deletions (indels) in the target genomic regions is generally regarded as a problem, as it introduces ambiguities in sequence alignments. However, we have recently shown that a high level of species discrimination is attainable in all taxa of life simply by considering the length of hypervariable regions defined by indel variants. Each species is tagged with a numeric profile of fragment lengths—a true numeric barcode. In this study, we describe a multifunctional computational workbench (named SPInDel for SPecies Identification by Insertions/Deletions) to assist researchers using variable‐length DNA sequences, and we demonstrate its applicability in molecular ecology. The SPInDel workbench provides a step‐by‐step environment for the alignment of target sequences, selection of informative hypervariable regions, design of PCR primers and the statistical validation of the species‐identification process. In our test data sets, we were able to discriminate all species from two genera of frogs (Ansonia and Leptobrachium) inhabiting lowland rainforests and mountain regions of South‐East Asia and species from the most common genus of coral reef fishes (Apogon). Our method can complement conventional DNA barcoding systems when indels are common (e.g. in rRNA genes) without the required step of DNA sequencing. The executable files, source code, documentation and test data sets are freely available at http://www.portugene.com/SPInDel/SPInDel_webworkbench.html .     

An improved method for utilizing high‐throughput amplicon sequencing to determine the diets of insectivorous animals

DNA analysis of predator faeces using high‐throughput amplicon sequencing (HTS) enhances our understanding of predator–prey interactions. However, conclusions drawn from this technique are constrained by biases that occur in multiple steps of the HTS workflow. To better characterize insectivorous animal diets, we used DNA from a diverse set of arthropods to assess PCR biases of commonly used and novel primer pairs for the mitochondrial gene, cytochrome oxidase C subunit 1 (COI). We compared diversity recovered from HTS of bat guano samples using a commonly used primer pair “ZBJ” to results using the novel primer pair “ANML.” To parameterize our bioinformatics pipeline, we created an arthropod mock community consisting of single‐copy (cloned) COI sequences. To examine biases associated with both PCR and HTS, mock community members were combined in equimolar amounts both pre‐ and post‐PCR. We validated our system using guano from bats fed known diets and using composite samples of morphologically identified insects collected in pitfall traps. In PCR tests, the ANML primer pair amplified 58 of 59 arthropod taxa (98%), whereas ZBJ amplified 24–40 of 59 taxa (41%–68%). Furthermore, in an HTS comparison of field‐collected samples, the ANML primers detected nearly fourfold more arthropod taxa than the ZBJ primers. The additional arthropods detected include medically and economically relevant insect groups such as mosquitoes. Results revealed biases at both the PCR and sequencing levels, demonstrating the pitfalls associated with using HTS read numbers as proxies for abundance. The use of an arthropod mock community allowed for improved bioinformatics pipeline parameterization.     

Multiplex restriction amplicon sequencing: a novel next‐generation sequencing‐based marker platform for high‐throughput genotyping

To enable rapid selection of traits in marker‐assisted breeding, markers must be technically simple, low‐cost, high‐throughput and randomly distributed in a genome. We developed such a technology, designated as Multiplex Restriction Amplicon Sequencing (MRASeq), which reduces genome complexity by polymerase chain reaction (PCR) amplification of amplicons flanked by restriction sites. The first PCR primers contain restriction site sequences at 3’‐ends, preceded by 6‐10 bases of specific or degenerate nucleotide sequences and then by a unique M13‐tail sequence which serves as a binding site for a second PCR that adds sequencing primers and barcodes to allow sample multiplexing for sequencing. The sequences of restriction sites and adjacent nucleotides can be altered to suit different species. Physical mapping of MRASeq SNPs from a biparental population of allohexaploid wheat (Triticum aestivum L.) showed a random distribution of SNPs across the genome. MRASeq generated thousands of SNPs from a wheat biparental population and natural populations of wheat and barley (Hordeum vulgare L.). This novel, next‐generation sequencing‐based genotyping platform can be used for linkage mapping to screen quantitative trait loci (QTL), background selection in breeding and many other genetics and breeding applications of various species.     

The Quantification of Representative Sequences pipeline for amplicon sequencing: case study on within‐population ITS1 sequence variation in a microparasite infecting Daphnia

Next generation sequencing (NGS) platforms are replacing traditional molecular biology protocols like cloning and Sanger sequencing. However, accuracy of NGS platforms has rarely been measured when quantifying relative frequencies of genotypes or taxa within populations. Here we developed a new bioinformatic pipeline (QRS) that pools similar sequence variants and estimates their frequencies in NGS data sets from populations or communities. We tested whether the estimated frequency of representative sequences, generated by 454 amplicon sequencing, differs significantly from that obtained by Sanger sequencing of cloned PCR products. This was performed by analysing sequence variation of the highly variable first internal transcribed spacer (ITS1) of the ichthyosporean Caullerya mesnili, a microparasite of cladocerans of the genus Daphnia. This analysis also serves as a case example of the usage of this pipeline to study within‐population variation. Additionally, a public Illumina data set was used to validate the pipeline on community‐level data. Overall, there was a good correspondence in absolute frequencies of C. mesnili ITS1 sequences obtained from Sanger and 454 platforms. Furthermore, analyses of molecular variance (amova ) revealed that population structure of C. mesnili differs across lakes and years independently of the sequencing platform. Our results support not only the usefulness of amplicon sequencing data for studies of within‐population structure but also the successful application of the QRS pipeline on Illumina‐generated data. The QRS pipeline is freely available together with its documentation under GNU Public Licence version 3 at http://code.google.com/p/quantification-representative-sequences .     

When to use next generation sequencing or diagnostic PCR in diet analyses

Next‐generation sequencing (NGS) is increasingly used for diet analyses; however, it may not always describe diet samples well. A reason for this is that diet samples contain mixtures of food DNA in different amounts as well as consumer DNA which can reduce the food DNA characterized. Because of this, detections will depend on the relative amount and identity of each type of DNA. For such samples, diagnostic PCR will most likely give more reliable results, as detection probability is only marginally dependent on other copresent DNA. We investigated the reliability of each method to test (a) whether predatory beetle regurgitates, supposed to be low in consumer DNA, allow to retrieve prey sequences using general barcoding primers that co‐amplify the consumer DNA, and (b) to assess the sequencing depth or replication needed for NGS and diagnostic PCR to give stable results. When consumer DNA is co‐amplified, NGS is better suited to discover the range of possible prey, than for comparing co‐occurrences of diet species between samples, as retested samples were repeatedly different in prey detections with this approach. This shows that samples were incompletely described, as prey detected by diagnostic PCR frequently were missed by NGS. As the sequencing depth needed to reliably describe the diet in such samples becomes very high, the cost‐efficiency and reliability of diagnostic PCR make diagnostic PCR better suited for testing large sample‐sets. Especially if the targeted prey taxa are thought to be of ecological importance, as diagnostic PCR gave more nested and consistent results in repeated testing of the same sample.