Publications by authors named "Kelly M Harkins"

10 Publications

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A novel NGS library preparation method to characterize native termini of fragmented DNA.

Nucleic Acids Res 2020 05;48(8):e47

Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA 95064, USA.

Biological and chemical DNA fragmentation generates DNA molecules with a variety of termini, including blunt ends and single-stranded overhangs. We have developed a Next Generation Sequencing (NGS) assay, XACTLY, to interrogate the termini of fragmented DNA, information traditionally lost in standard NGS library preparation methods. Here we describe the XACTLY method, showcase its sensitivity and specificity, and demonstrate its utility in in vitro experiments. The XACTLY assay is able to report relative abundances of all lengths and types (5' and 3') of single-stranded overhangs, if present, on each DNA fragment with an overall accuracy between 80-90%. In addition, XACTLY retains the sequence of each native DNA molecule after fragmentation and can capture the genomic landscape of cleavage events at single nucleotide resolution. The XACTLY assay can be applied as a novel research and discovery tool for fragmentation analyses and in cell-free DNA.
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http://dx.doi.org/10.1093/nar/gkaa128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192605PMC
May 2020

A ligation-based single-stranded library preparation method to analyze cell-free DNA and synthetic oligos.

BMC Genomics 2019 Dec 27;20(1):1023. Epub 2019 Dec 27.

Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA, 95064, USA.

Background: Cell-free DNA (cfDNA), present in circulating blood plasma, contains information about prenatal health, organ transplant reception, and cancer presence and progression. Originally developed for the genomic analysis of highly degraded ancient DNA, single-stranded DNA (ssDNA) library preparation methods are gaining popularity in the field of cfDNA analysis due to their efficiency and ability to convert short, fragmented DNA into sequencing libraries without altering DNA ends. However, current ssDNA methods are costly and time-consuming.

Results: Here we present an efficient ligation-based single-stranded library preparation method that is engineered to produce complex libraries in under 2.5 h from as little as 1 nanogram of input DNA without alteration to the native ends of template molecules. Our method, called Single Reaction Single-stranded LibrarY or SRSLY, ligates uniquely designed Next-Generation Sequencing (NGS) adapters in a one-step combined phosphorylation/ligation reaction that foregoes end-polishing. Using synthetic DNA oligos and cfDNA, we demonstrate the efficiency and utility of this approach and compare with existing double-stranded and single-stranded approaches for library generation. Finally, we demonstrate that cfDNA NGS data generated from SRSLY can be used to analyze DNA fragmentation patterns to deduce nucleosome positioning and transcription factor binding.

Conclusions: SRSLY is a versatile tool for converting short and fragmented DNA molecules, like cfDNA fragments, into sequencing libraries while retaining native lengths and ends.
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http://dx.doi.org/10.1186/s12864-019-6355-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935139PMC
December 2019

Genetic Ancestry of Rapanui before and after European Contact.

Curr Biol 2017 Oct 12;27(20):3209-3215.e6. Epub 2017 Oct 12.

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.

The origins and lifeways of the inhabitants of Rapa Nui (Easter Island), a remote island in the southeast Pacific Ocean, have been debated for generations. Archaeological evidence substantiates the widely accepted view that the island was first settled by people of Polynesian origin, as late as 1200 CE [1-4]. What remains controversial, however, is the nature of events in the island's population history prior to the first historic contact with Europeans in 1722 CE. Purported contact between Rapa Nui and South America is particularly contentious, and recent studies have reported genetic evidence for Native American admixture in present-day indigenous inhabitants of Rapa Nui [5-8]. Statistical modeling has suggested that this genetic contribution might have occurred prior to European contact [6]. Here we directly test the hypothesis that the Native American admixture of the current Rapa Nui population predates the arrival of Europeans with a paleogenomic analysis of five individual samples excavated from Ahu Nau Nau, Anakena, dating to pre- and post-European contact, respectively. Complete mitochondrial genomes and low-coverage autosomal genomes show that the analyzed individuals fall within the genetic diversity of present-day and ancient Polynesians, and we can reject the hypothesis that any of these individuals had substantial Native American ancestry. Our data thus suggest that the Native American ancestry in contemporary Easter Islanders was not present on the island prior to European contact and may thus be due to events in more recent history.
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http://dx.doi.org/10.1016/j.cub.2017.09.029DOI Listing
October 2017

Phylogenomic reconstruction supports supercontinent origins for Leishmania.

Infect Genet Evol 2016 Mar 18;38:101-109. Epub 2015 Dec 18.

School of Human Evolution and Social Change, Arizona State University, Tempe, AZ, USA; Center for Evolution and Medicine, Arizona State University, Tempe, AZ, USA.

Leishmania, a genus of parasites transmitted to human hosts and mammalian/reptilian reservoirs by an insect vector, is the causative agent of the human disease complex leishmaniasis. The evolutionary relationships within the genus Leishmania and its origins are the source of ongoing debate, reflected in conflicting phylogenetic and biogeographic reconstructions. This study employs a recently described bioinformatics method, SISRS, to identify over 200,000 informative sites across the genome from newly sequenced and publicly available Leishmania data. This dataset is used to reconstruct the evolutionary relationships of this genus. Additionally, we constructed a large multi-gene dataset, using it to reconstruct the phylogeny and estimate divergence dates for species. We conclude that the genus Leishmania evolved at least 90-100 million years ago, supporting a modified version of the Multiple Origins hypothesis that we call the Supercontinent hypothesis. According to this scenario, separate Leishmania clades emerged prior to, and during, the breakup of Gondwana. Additionally, we confirm that reptile-infecting Leishmania are derived from mammalian forms and that the species that infect porcupines and sloths form a clade long separated from other species. Finally, we firmly place the guinea-pig infecting species, Leishmaniaenriettii, the globally dispersed Leishmaniasiamensis, and the newly identified Australian species from a kangaroo, as sibling species whose distribution arises from the ancient connection between Australia, Antarctica, and South America.
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http://dx.doi.org/10.1016/j.meegid.2015.11.030DOI Listing
March 2016

Pre-Columbian tuberculosis in Tierra del Fuego? Discussion of the paleopathological and molecular evidence.

Int J Paleopathol 2015 Dec 17;11:92-101. Epub 2015 Nov 17.

Instituto de Bioantropología, Museo de la Naturaleza y el Hombre, Tenerife, Islas Canarias, Spain.

This work contributes to ongoing discussions about the nature of tuberculosis in the Western Hemisphere prior to the time of European contact. Our example, from the extreme south of South America was, at the time of our study, without firm temporal association or molecular characterization. In Tierra del Fuego, Constantinescu (1999) briefly described vertebral bone lesions compatible with TB in an undated skeleton from Myren 1 site (Chile). The remains of Myren are estimated to represent a man between 18 and 23 years old at the time of death. The objectives of this research are to extend this description, to present molecular results, to establish a radiocarbon date, and to report stable isotopic values for the remains. We provide further description of the remains, including tuberculosis-like skeletal pathology. Radiocarbon dating of 640±20 years BP attributes this individual to the precontact fourteenth-fifteenth centuries. Isotopic ratios for nitrogen and carbon from bone collagen suggest a mixed diet. Molecular results were positive for the rpoB quantitative PCR (qPCR) assays but negative for two independent IS6110 and IS1081 qPCR assays. Further testing using genomic methods to target any mycobacteria for specific identification are needed.
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http://dx.doi.org/10.1016/j.ijpp.2015.09.003DOI Listing
December 2015

A composite genome approach to identify phylogenetically informative data from next-generation sequencing.

BMC Bioinformatics 2015 Jun 11;16:193. Epub 2015 Jun 11.

The Biodesign Institute, Arizona State University, Tempe, AZ, USA.

Background: Improvements in sequencing technology now allow easy acquisition of large datasets; however, analyzing these data for phylogenetics can be challenging. We have developed a novel method to rapidly obtain homologous genomic data for phylogenetics directly from next-generation sequencing reads without the use of a reference genome. This software, called SISRS, avoids the time consuming steps of de novo whole genome assembly, multiple genome alignment, and annotation.

Results: For simulations SISRS is able to identify large numbers of loci containing variable sites with phylogenetic signal. For genomic data from apes, SISRS identified thousands of variable sites, from which we produced an accurate phylogeny. Finally, we used SISRS to identify phylogenetic markers that we used to estimate the phylogeny of placental mammals. We recovered eight phylogenies that resolved the basal relationships among mammals using datasets with different levels of missing data. The three alternate resolutions of the basal relationships are consistent with the major hypotheses for the relationships among mammals, all of which have been supported previously by different molecular datasets.

Conclusions: SISRS has the potential to transform phylogenetic research. This method eliminates the need for expensive marker development in many studies by using whole genome shotgun sequence data directly. SISRS is open source and freely available at https://github.com/rachelss/SISRS/releases.
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http://dx.doi.org/10.1186/s12859-015-0632-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4464851PMC
June 2015

Ancient pathogen genomics: insights into timing and adaptation.

J Hum Evol 2015 Feb 18;79:137-49. Epub 2014 Dec 18.

Center for Bioarchaeological Research, School of Human Evolution and Social Change, Arizona State University, United States.

Disease is a major cause of natural selection affecting human evolution, whether through a sudden pandemic or persistent morbidity and mortality. Recent contributions in the field of ancient pathogen genomics have advanced our understanding of the antiquity and nature of human-pathogen interactions through time. Technical advancements have facilitated the recovery, enrichment, and high-throughput sequencing of pathogen and parasite DNA from archived and archaeological remains. These time-stamped genomes are crucial for calibrating molecular clocks to infer the timing of evolutionary events, while providing finer-grain resolution to phylogenetic reconstructions and complex biogeographical patterns. Additionally, genome scale data allow better identification of substitutions linked to adaptations of the pathogen to their human hosts. As methodology continues to improve, ancient genomes of humans and their diverse microbiomes from a range of eras and archaeological contexts will enable population-level ancient analyses in the near future and a better understanding of their co-evolutionary history.
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http://dx.doi.org/10.1016/j.jhevol.2014.11.002DOI Listing
February 2015

Screening ancient tuberculosis with qPCR: challenges and opportunities.

Philos Trans R Soc Lond B Biol Sci 2015 Jan;370(1660):20130622

School of Human Evolution and Social Change, Arizona State University, Tempe, AZ, USA

The field of ancient DNA (aDNA) has rapidly accelerated in recent years as a result of new methods in next-generation sequencing, library preparation and targeted enrichment. Such research is restricted, however, by the highly variable DNA preservation within different tissues, especially when isolating ancient pathogens from human remains. Identifying positive candidate samples via quantitative PCR (qPCR) for downstream procedures can reduce reagent costs, increase capture efficiency and maximize the number of sequencing reads of the target. This study uses four qPCR assays designed to target regions within the Mycobacterium tuberculosis complex (MTBC) to examine 133 human skeletal samples from a wide geographical and temporal range, identified by the presence of skeletal lesions typical of chronic disseminated tuberculosis. Given the inherent challenges working with ancient mycobacteria, strict criteria must be used and primer/probe design continually re-evaluated as new data from bacteria become available. Seven samples tested positive for multiple MTBC loci, supporting them as strong candidates for downstream analyses. Using strict and conservative criteria, qPCR remains a fast and effective screening tool when compared with screening by more expensive sequencing and enrichment technologies.
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http://dx.doi.org/10.1098/rstb.2013.0622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4275897PMC
January 2015

Pre-Columbian mycobacterial genomes reveal seals as a source of New World human tuberculosis.

Nature 2014 Oct 20;514(7523):494-7. Epub 2014 Aug 20.

1] Department of Archaeological Sciences, University of Tübingen, Ruemelinstraße 23, 72070 Tübingen, Germany [2] Senckenberg Centre for Human Evolution and Palaeoenvironment, University of Tübingen, Tübingen 72070, Germany [3] Max Planck Institute for Science and History, Khalaische Straße 10, 07745 Jena, Germany.

Modern strains of Mycobacterium tuberculosis from the Americas are closely related to those from Europe, supporting the assumption that human tuberculosis was introduced post-contact. This notion, however, is incompatible with archaeological evidence of pre-contact tuberculosis in the New World. Comparative genomics of modern isolates suggests that M. tuberculosis attained its worldwide distribution following human dispersals out of Africa during the Pleistocene epoch, although this has yet to be confirmed with ancient calibration points. Here we present three 1,000-year-old mycobacterial genomes from Peruvian human skeletons, revealing that a member of the M. tuberculosis complex caused human disease before contact. The ancient strains are distinct from known human-adapted forms and are most closely related to those adapted to seals and sea lions. Two independent dating approaches suggest a most recent common ancestor for the M. tuberculosis complex less than 6,000 years ago, which supports a Holocene dispersal of the disease. Our results implicate sea mammals as having played a role in transmitting the disease to humans across the ocean.
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http://dx.doi.org/10.1038/nature13591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4550673PMC
October 2014

Effects of 2 ankle fatigue models on the duration of postural stability dysfunction.

J Athl Train 2005 Jul-Sep;40(3):191-4

University of Kentucky, Lexington, KY 40536-0200, USA.

Context: Muscle fatigue is generally categorized in 2 ways: that caused by peripheral weakness (peripheral fatigue) and that caused by a progressive failure of voluntary neural drive (central fatigue). Numerous variables have been studied in conjunction with fatigue protocols, including postural stability, maximum voluntary contraction force, and reaction time. When torque recordings fall below 50% of a maximum voluntary contraction, the muscle is described as fatigued, but whether this value is a good indicator of fatigue has not been studied.

Objective: To compare the effects of 2 ankle musculature fatigue protocols (30% and 50%) on the duration of postural stability dysfunction.

Design: To assess differences between the 30% and 50% fatigue protocols, we calculated a 1 between-groups factor (subjects) and 2 within-groups factors (fatigue, test) analysis of variance.

Setting: E.J. Nutter Athletic Training Facility.

Patients Or Other Participants: Twenty subjects (10 men, 10 women; age = 21.15 +/- 2.23 years; height = 172.97 +/- 9.86 cm; mass = 70.62 +/- 14.60 kg) volunteered for this study. Subjects had no history of lower extremity injury, vestibular or balance disorders, functional ankle instability, or head injury in the past 6 months.

Intervention(s): On separate days, subjects performed isokinetic fatiguing contractions of the plantar flexors and dorsiflexors in a 30% protocol (70% decrease in strength) and a 50% protocol (50% decrease in strength).

Main Outcome Measure(s): Baseline and postfatigue postural stability scores were determined before and after the isokinetic fatiguing contractions. Plantar-flexion peak-torque measurements were obtained for the 2 fatiguing protocols. Three prefatigue and 12 postfatigue postural stability trials were recorded. Velocities for testing were 60 degrees /s for plantar flexion and 120 degrees /s for dorsiflexion.

Results: Sway velocity was significantly greater when the ankle was fatigued to 30% (1.56 degrees /s) than in the 50% condition (1.36 degrees /s). For the 30% protocol, sway was significantly impaired when the pretest condition (1.19 degrees /s) was compared with posttest trial 1 (2.34 degrees /s), trial 2 (2.37 degrees /s), and trial 3 (1.71 degrees /s). For the 50% protocol, sway was significantly impaired when the pretest condition (1.27 degrees /s) was compared with posttest trial 1 (2.02 degrees /s).

Conclusions: The 30% fatigue protocol resulted in significantly longer impairment of postural stability than the 50% protocol. Because the 30% protocol resulted in a greater effect but was relatively short-lived (approximately 75 to 90 s), it is more useful for research purposes.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1250260PMC
June 2007