A customised target capture sequencing tool for molecular identification of Aloe vera and relatives

Plant molecular identification studies have, until recently, been limited to the use of highly conserved markers from plastid and other organellar genomes, compromising resolution in highly diverse plant clades. Due to their higher evolutionary rates and reduced paralogy, low-copy nuclear genes overcome this limitation but are difficult to sequence with conventional methods and require high-quality input DNA. Aloe vera and its relatives in the Alooideae clade (Asphodelaceae, subfamily Asphodeloideae) are of economic interest for food and health products and have horticultural value. However, pressing conservation issues are increasing the need for a molecular identification tool to regulate the trade. With > 600 species and an origin of ± 15 million years ago, this predominantly African succulent plant clade is a diverse and taxonomically complex group for which low-copy nuclear genes would be desirable for accurate species discrimination. Unfortunately, with an average genome size of 16.76 pg, obtaining high coverage sequencing data for these genes would be prohibitively costly and computationally demanding. We used newly generated transcriptome data to design a customised RNA-bait panel targeting 189 low-copy nuclear genes in Alooideae. We demonstrate its efficacy in obtaining high-coverage sequence data for the target loci on Illumina sequencing platforms, including degraded DNA samples from museum specimens, with considerably improved phylogenetic resolution. This customised target capture sequencing protocol has the potential to confidently indicate phylogenetic relationships of Aloe vera and related species, as well as aid molecular identification applications.

Authentication of garlic (Allium sativum L.) supplements using a trnLUAA mini-barcode

Garlic (Allium sativum), a widely distributed plant with great cultural and medicinal significance, is one of the most popular herbal dietary supplements in Europe and North America. Garlic supplements are consumed for a variety of reasons, including for their purported antihypertensive, antibacterial, and anticarcinogenic effects. The steady increase in the global herbal dietary supplement market paired with a global patchwork of regulatory frameworks makes the development of assays for authentication of these products increasingly important. A DNA mini-barcode assay was developed using the P6 loop of the plastid trnLUAA intron to positively identify A. sativum products. Analysis of 43 commercially available garlic herbal dietary supplements produced mini-barcode sequences for 33 supplements, all of which contained detectable amounts of A. sativum. The trnLUAA P6 mini-barcode can be highly useful for specimen identification, particularly for samples that may contain degraded DNA.

Molecular Diagnosis of T-cell Lymphoma: A correlative study of PCR-based T-cell clonality assessment and targeted NGS

Immunomorphological diagnosis of T-cell lymphomas (TCL) may be challenging, especially on needle biopsies. Multiplex polymerase chain reaction (PCR) assays to assess T-cell receptor (TCR) gene rearrangements are now widely used to detect T-cell clones and provide diagnostic support. However, PCR assays only detect 80% of TCL, and clonal lymphocyte populations may also appear in non-neoplastic conditions. More recently, targeted next-generation sequencing (t-NGS) technologies have been deployed to improve lymphoma classification. To the best of our knowledge, the comparison of these techniques' performance in TCL diagnosis has not been reported yet. In this study, 82 TCL samples and 25 non-neoplastic T-cell infiltrates were divided into two cohorts (test and validation) and analyzed with both multiplex PCR and t-NGS to investigate TCR gene rearrangements and somatic mutations, respectively. The detection of mutations appeared to be more specific (100.0%) than T-cell clonality assessment (41.7% to 45.5%), whereas no differences were observed in terms of sensitivity (95.1% to 97.4%). Furthermore, t-NGS provided a reliable basis for TCL diagnosis in samples with partially degraded DNA that was impossible to assess with PCR. Finally, although multiplex PCR assays appeared to be less specific than t-NGS, both techniques remain complementary, as PCR recovered some t-NGS negative cases.

Systematics of the Arboreal Neotropical ‘thorellii’ Clade of Centruroides Bark Scorpions (Buthidae) and the Efficacy of Mini-Barcodes for Museum Specimens

Fragmented and degraded DNA is pervasive among museum specimens, hindering molecular phylogenetics and species identification. Mini-barcodes, 200–300-base-pair (bp) fragments of barcoding genes, have proven effective for species-level identification of specimens from which complete barcodes cannot be obtained in many groups, but have yet to be tested in arachnids. The present study investigated the efficacy of mini-barcodes combined with longer sequences of the Cytochrome c Oxidase Subunit I (COI) gene in the systematics of the arboreal Neotropical ‘thorellii’ clade of Centruroides Marx, 1890 bark scorpions (Buthidae, C.L. Koch 1837), the species of which have proven to be difficult to identify and delimit due to their similar morphology. The phylogeny of 53 terminals, representing all nine species of the clade and representative species belonging to related clades of Centruroides, rooted on Heteroctenus junceus (Herbst, 1800) and based on up to 1078 base pairs of COI and 112 morphological characters, is presented to test the monophyly of the clade and the limits of its component species. The results support the recognition of nine species of the ‘thorellii’ clade, in accordance with a recent taxonomic revision, and highlight the efficacy of mini-barcodes for identifying morphologically similar cryptic species using specimens of variable age and preservation.

Reverse complement-PCR, an innovative and effective method for multiplexing forensically relevant single nucleotide polymorphism marker systems

DNA analyses from challenging samples such as touch evidence, hairs and skeletal remains push the limits of the current forensic DNA typing technologies. Reverse complement PCR (RC-PCR) is a novel, single-step PCR target enrichment method adapted to amplify degraded DNA. The sample preparation process involves a limited number of steps, decreasing the labor required for library preparation and reducing the possibility of contamination due to less sample manipulation. These features of the RC-PCR make the technology a unique application to successfully target single nucleotide polymorphisms (SNPs) in fragmented and low copy number DNA and yield results from samples in which no or limited data are obtained with standard DNA typing methods. The developed RC-PCR short amplicon 85 SNP-plex panel is a substantial improvement over the previously reported 27-plex RC-PCR multiplex that will provide higher discrimination power for challenging DNA sample analyses.

Evaluation of a high-throughput, cost-effective Illumina library preparation kit

Library preparation for high-throughput sequencing applications is a critical step in producing representative, unbiased sequencing data. The iGenomX Riptide High Throughput Rapid Library Prep Kit purports to provide high-quality sequencing data with lower costs compared to other Illumina library kits. To test these claims, we compared sequence data quality of Riptide libraries to libraries constructed with KAPA Hyper and NEBNext Ultra. Across several single-source genome samples, mapping performance and de novo assembly of Riptide libraries were similar to conventional libraries prepared with the same DNA. Poor performance of some libraries resulted in low sequencing depth. In particular, degraded DNA samples may be challenging to sequence with Riptide. There was little cross-well plate contamination with the overwhelming majority of reads belong to the proper source genomes. The sequencing of metagenome samples using different Riptide primer sets resulted in variable taxonomic assignment of reads. Increased adoption of the Riptide kit will decrease library preparation costs. However, this method might not be suitable for degraded DNA.

Sixteenth-century tomatoes in Europe: who saw them, what did they look like, and where did they come from?

BackgroundSoon after the Spanish conquest of the Americas, the first tomatoes were presented as curiosities to the European royals and drew the attention of sixteenth-century Italian naturalists. Despite of their scientific interest in this New World crop, most Renaissance botanists did not specify where these ‘golden apples’ or ‘pomi d’oro’ came from. The debate on the first European tomatoes and their origin is often hindered by erroneous dating, botanical misidentifications and inaccessible historical sources. The discovery of a tomato specimen in the sixteenth-century ‘En Tibi herbarium’ kept at Leiden, the Netherlands led to claims that its DNA would reveal the ‘original’ taste and pest resistance of early tomatoes.MethodsRecent digitization efforts greatly facilitate research on historic botanical sources. Here we provide an overview of the ten remaining sixteenth-century tomato specimens, early descriptions and 13 illustrations. Several were never published before, revealing what these tomatoes looked like, who saw them, and where they came from.ResultsOur survey shows that the earliest tomatoes in Europe came in a much wider variety of colors, shapes and sizes than previously thought, with both simple and fasciated flowers, round and segmented fruits. Pietro Andrea Matthioli gave the first description of a tomato in 1544, and the oldest specimens were collected by Ulisse Aldrovandi and Francesco Petrollini in c. 1551 from plants grown in the Pisa botanical garden by their teacher Luca Ghini. The oldest illustrations were made in Germany in the early 1550s, but the Flemish Rembert Dodoens published the first image in 1553. The names of early tomatoes in contemporary manuscripts suggest both a Mexican and a Peruvian origin. The ‘En Tibi’ specimen was collected by Petrollini around Bologna in 1558 and thus is not the oldest extant tomato. Although only 1.2% of its DNA was readable, recent molecular research shows that the En Tibi tomato was a fully domesticated, but quite heterozygous individual and genetically close to three Mexican and two Peruvian tomato landraces. Molecular research on the other sixteenth-century tomato specimens may reveal other patterns of genetic similarity and geographic origin. Clues on the ‘historic’ taste and pest resistance of the sixteenth-century tomatoes should not be searched in their degraded DNA, but rather in those landraces in Central and South America that are genetically close to them. The indigenous farmers growing these traditional varieties should be supported to conserve these heirloom varieties in-situ.

The Effects of Herbarium Specimen Characteristics on Short-Read NGS Sequencing Success in Nearly 8000 Specimens: Old, Degraded Samples Have Lower DNA Yields but Consistent Sequencing Success

Phylogenetic datasets are now commonly generated using short-read sequencing technologies unhampered by degraded DNA, such as that often extracted from herbarium specimens. The compatibility of these methods with herbarium specimens has precipitated an increase in broad sampling of herbarium specimens for inclusion in phylogenetic studies. Understanding which sample characteristics are predictive of sequencing success can guide researchers in the selection of tissues and specimens most likely to yield good results. Multiple recent studies have considered the relationship between sample characteristics and DNA yield and sequence capture success. Here we report an analysis of the relationship between sample characteristics and sequencing success for nearly 8,000 herbarium specimens. This study, the largest of its kind, is also the first to include a measure of specimen quality (“greenness”) as a predictor of DNA sequencing success. We found that taxonomic group and source herbarium are strong predictors of both DNA yield and sequencing success and that the most important specimen characteristics for predicting success differ for DNA yield and sequencing: greenness was the strongest predictor of DNA yield, and age was the strongest predictor of proportion-on-target reads recovered. Surprisingly, the relationship between age and proportion-on-target reads is the inverse of expectations; older specimens performed slightly better in our capture-based protocols. We also found that DNA yield itself is not a strong predictor of sequencing success. Most literature on DNA sequencing from herbarium specimens considers specimen selection for optimal DNA extraction success, which we find to be an inappropriate metric for predicting success using next-generation sequencing technologies.

A Customised Target Capture Sequencing Tool for Molecular Identification of Aloe Vera and Relatives

Plant molecular identification studies have, until recently, been limited to the use of highly conserved markers from plastid and other organellar genomes, compromising resolution in highly diverse plant clades. Due to their higher evolutionary rates and reduced paralogy, low-copy nuclear genes overcome this limitation but are difficult to sequence with conventional methods and require high-quality input DNA. Aloe vera and its relatives (Asphodelaceae, subfamily Alooideae) are of economic interest for food and health products and have horticultural value. However, pressing conservation issues are increasing the need for a molecular identification tool to regulate the trade. With >600 species and an origin of ±15 million years ago, this predominantly African succulent plant clade is a diverse and taxonomically complex group for which low-copy nuclear genes would be desirable for accurate species discrimination. Unfortunately, with an average genome size of 16.76 pg, obtaining high coverage sequencing data for these genes would be prohibitively costly and computationally demanding. We used newly generated transcriptome data to design a customised RNA-bait panel targeting 189 low-copy nuclear genes in Alooideae. We demonstrate its efficacy in obtaining high-coverage sequence data for the target loci on Illumina sequencing platforms, including degraded DNA samples from museum specimens, with considerably improved phylogenetic resolution. This customised target capture sequencing protocol has the potential to confidently indicate phylogenetic relationships of Aloe vera and related species, as well as aid molecular identification applications.