Acct 301 Week 5 Homework Problem 2038

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Next-generation-sequencing (NGS) technologies combined with a classic DNA barcoding approach have enabled fast and credible measurement for biodiversity of mixed environmental samples. However, the PCR amplification involved in nearly all existing NGS protocols inevitably introduces taxonomic biases. In the present study, we developed new Illumina pipelines without PCR amplifications to analyze terrestrial arthropod communities.


Mitochondrial enrichment directly followed by Illumina shotgun sequencing, at an ultra-high sequence volume, enabled the recovery of Cytochrome c Oxidase subunit 1 (COI) barcode sequences, which allowed for the estimation of species composition at high fidelity for a terrestrial insect community. With 15.5 Gbp Illumina data, approximately 97% and 92% were detected out of the 37 input Operational Taxonomic Units (OTUs), whether the reference barcode library was used or not, respectively, while only 1 novel OTU was found for the latter. Additionally, relatively strong correlation between the sequencing volume and the total biomass was observed for species from the bulk sample, suggesting a potential solution to reveal relative abundance.


The ability of the new Illumina PCR-free pipeline for DNA metabarcoding to detect small arthropod specimens and its tendency to avoid most, if not all, false positives suggests its great potential in biodiversity-related surveillance, such as in biomonitoring programs. However, further improvement for mitochondrial enrichment is likely needed for the application of the new pipeline in analyzing arthropod communities at higher diversity.


Given the increasing needs for assessing habitat quality and conserving natural bio-resources, biodiversity composition and its temporal and spatial variations have been evaluated systematically using standardized protocols [1,2]. National biomonitoring programs have been established globally, such as in the United States, United Kingdom, Australia and Canada [3–6]. Although specific protocols and sampling scales vary across nations, tens of thousands of sampling sites are collected multiple times a year, through which millions of biological specimens are routinely collected, preserved, identified and statistically analyzed [7]. This biological information is used by environmental agencies as the scientific basis for decision-making [2,3,5,8]. However, the major impediment to this application has been the limited capacities in morphological identification of taxonomic diversity for large volumes of biological samples in an accurate and high-throughput manner [9]. Although many diversity analyses employed in biological assessments are of high-quality and credibility, the limited identification capacity may lead to coarse diversity resolution [10], inconsistent taxonomic delineation across individual researchers and institutes [11], much reduced sampling scale [7], and extended turn-around time in routine sample processes [12].

DNA barcoding, which utilizes a standard gene fragment for species identification, has been widely used to facilitate biodiversity and ecological studies [13]. When the classic DNA barcoding approach, which is optimized based on individual Sanger sequencing, is coupled with next-generation-sequencing (NGS) technologies, the combined method, metabarcoding [14], shows even greater potential in unveiling molecular characteristics of the entire fauna or flora in question. In particular, metabarcoding has enabled sophisticated analyses of biodiversity in varied environments, ranging from deep-sea meiofauna [15] to terrestrial insects collected by Malaise traps [16], while the majority of studies have focused on microbial communities [17,18]. In addition to the ability to reveal diversity for mixed biological samples, NGS platforms are capable of high-throughput sequencing [19,20] with short turnaround time (e.g., 24 hours for the Illumina MiSeq and Roche 454 GS FLX + sequencers).

In nearly all published works, NGS analyses of biodiversity usually involve DNA extraction of bulk samples (mixtures of co-occurring taxonomic groups), PCR amplification of targeted genetic markers, and NGS analysis for taxonomic composition (Figure 1). PCR amplification of targeted genes is employed as the sole approach to acquiring sufficient barcode sequences that are used for species identification. An inherent drawback to this approach is that primers designed to amplify the full range of taxa presented in the bulk sample are rarely “universal”, with different amplification efficiencies in varied organisms [21–28]. Although much effort has been made to increase the universality of primer sets [29–32], it is difficult to predict the performance of primers when the investigated fauna is largely unknown. As a result, it seems to be impossible to completely eliminate the taxonomic biases introduced by PCR. In addition, our recent work (unpublished data) found that amplification errors (such as mismatches to the template DNA) propagated during PCR could be readily detected by the highly sensitive NGS technology, therefore increasing the ratio of false positives — which is potentially one of the major causes of what is commonly known as “biodiversity inflation” or “false positives” found in nearly all published NGS analyses of biodiversity [16,33]. Furthermore, the success of PCR amplifications is also influenced by the nucleotide composition and secondary structure of the DNA templates. For instance, homopolymers — a long strain of identical nucleotides arranged in tandem, present a challenge for the polymerase to pass through. If these nucleotide characteristics are taxon specific, amplification efficiency will create taxonomic biases despite primer optimization. The DNA barcoding of subgroups of Hymenoptera presents a notoriously difficult example where poly-Ts are commonly found in regions of the Cytochrome c Oxidase subunit 1 gene (COI) barcodes. This characteristic has led to low success in the acquisition of both full-length barcodes [34,35] and in taxonomic detection for a large portion of Hymenoptera in recent NGS biodiversity analyses [16]. Lastly, once PCR is included in the NGS analytical pipeline, abundance information of each of the member taxon in the community will be inevitably lost. Although some correlation of NGS reads and taxonomic abundance has been shown in mixed nematode samples [36] and diet analyses [37,38], such success will largely depend on the phylogenetic diversity of taxa in question and the performance of primers applied, which is challenging for most animal groups used as biological proxies (e.g., macroinvertebrates). Protocols such as DNA capture and environmental DNA shotgun sequencing have been proposed by Taberlet et al. [39] to bypass PCR. Some preliminary work also showed the feasibility of applying this approach on eukaryotic diversity [40]. But systematic metabarcoding studies on real eukaryotic communities that are independent of PCR are lacking.

In this study, we aim to develop a new NGS pipeline that is independent of PCR amplifications (Figure 1), while still enabling molecular identification of insects at the species level, using bulk insect samples for the proof of concept. Two major challenges for the complete elimination of PCR amplification need to be resolved first: (a) detection of target DNA sequences at low quantity and (b) taxonomic assignment based on short NGS reads. A 650 bp sequence fragment on the 5′ end of the mtCOI gene has been widely adopted as the DNA barcode region for identifying animal species since its initial proposal [13]. Although mitochondria are found in vast copy numbers in metazoan animals, mitochondria (MT) nucleotides only account for a small fraction of the total DNA compared to nuclear sequences (e.g., 0.05% in Bombyx mori [41]) and sequences of microbial origin in the DNA soup. The ultra-deep sequencing capacity of the Illumina sequencing platform provides an opportunity to examine the feasibility of detecting minute trace of mitochondrial sequences directly from genomic DNA mixtures. For example, each sequencing run of the HiSeq 2000 sequencer is able to produce approximately 600 Gbp, equivalent to 200 human genomes, which is more than 1,000X as much as the capacity of the Roche 454 GS FLX + platform (see review [20]). On the other hand, the current Illumina sequence reads for HiSeq 2000 can only reach up to 150 bp. This short sequence length presents a limitation to the full utilization of full-length DNA barcodes (approximately 650 bp for animal COI barcodes) available globally (e.g., through the International Barcode of Life initiative [42]). Although a very short piece of the standard barcodes (“mini-barcode”) of only 130 bp demonstrated reliable taxonomic resolution for a number of animal groups [43], longer sequences are always preferred for improved identification power. Therefore, we need informatics solutions to assign species-level identity based on mixed short Illumina reads.

To overcome these two major hurdles, the new pipeline employed in the current study involves pre-sequencing enrichment of mitochondria followed by total DNA extraction, shotgun sequencing of isolated total DNA using the Illumina HiSeq 2000, and taxonomic identification of NGS reads. Two different approaches can be applied to assign species-level identity: (1) if an a priori barcode reference library exists for the investigated fauna, NGS reads are mapped to the reference sequences following defined criteria; and (2) when this reference library is absent, de novo assembly of NGS reads into mitochondrial gene fragments, especially the COI barcode region, is employed, followed by gene annotation, to ensure accurate detection of taxa from the mixed bulk sample. Based on the promising results from our in silico simulations using 209 insect mitochondrial genomes obtained from GenBank (details of the design and results of the simulation are provided in Additional file 1: Appendix S1), we apply the new Illumina pipeline to analyze real bulk insect samples. A preliminary study was first performed to gain a basic understanding for the required scale of Illumina sequencing. Details were summarized in Additional file 2: Appendix S2. A formal sample was subsequently sequenced and analyzed with ultra-high sequencing volume (15.5 Gb) to systematically test and discuss this NGS pipeline on biodiversity study in the following text.

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