Optimization of capture protocols across species targeting up to 32000 genes and their extension to pooled DNA

In-solution based capture is becoming a method of choice for sequencing targeted sequence. We assessed and optimized a capture protocol in 20 different species from 6 different plant genus using kits from 20,000 to 200,000 baits targeting from 300 to 32,000 genes. We evaluated both the effectiveness of the capture protocol and the fold enrichment in targeted sequences. We proposed a protocol with multiplexing up to 96 samples in a single hybridization and showed it was an efficient and cost-effective strategy. We also extended the use of capture to pools of 100 samples and proved the efficiency of the method to assess allele frequency. Using a set of various organisms with different genome sizes, we demonstrated a correlation between the percentage of on-target reads vs. the relative size of the targeted sequences. Altogether, we proposed methods, strategies, cost-efficient protocols and statistics to better evaluate and more effectively use hybridization capture.

Towards harmonization of DNA metabarcoding for monitoring marine macrobenthos: the effect of technical replicates and pooled DNA extractions on species detection

DNA-based monitoring methods are potentially faster and cheaper compared to traditional morphological benthic identification. DNA metabarcoding involves various methodological choices which can introduce bias leading to a different outcome in biodiversity patterns. Therefore, it is important to harmonize DNA metabarcoding protocols to allow comparison across studies and this requires a good understanding of the effect of methodological choices on diversity estimates. This study investigated the impact of DNA and PCR replicates on the detection of macrobenthos species in locations with high, medium and low diversity. Our results show that two to three DNA replicates were needed in locations with a high and medium diversity to detect at least 80% of the species found in the six DNA replicates, while three to four replicates were needed in the location with low diversity. In contrast to general belief, larger body size or higher abundance of the species in a sample did not increase its detection prevalence among DNA replicates. However, rare species were less consistently detected across all DNA replicates of the location with high diversity compared to locations with less diversity. Our results further show that pooling of DNA replicates did not significantly alter diversity patterns, although a small number of rare species was lost. Finally, our results confirm high variation in species detection between PCR replicates, especially for the detection of rare species. These results contribute to create reliable, time and cost efficient metabarcoding protocols for the characterization of macrobenthos.

Identification of Tick-Borne Pathogens and Genotyping of Coxiella burnetii in Rhipicephalus microplus in Yunnan Province, China

Rhipicephalus microplus, a vector that can transmit many pathogens to humans and domestic animals, is widely distributed in Yunnan province, China. However, few reports on the prevalence of tick-borne pathogens (TBPs) in Rh. microplus in Yunnan are available. The aim of this study was to detect TBPs in Rh. microplus in Yunnan and to analyze the phylogenetic characterization of TBPs detected in these ticks. The adult Rh. microplus (n = 516) feeding on cattle were collected. The pooled DNA samples of these ticks were evaluated using metagenomic next-generation sequencing (mNGS) and then TBPs in individual ticks were identified using genus- or group-specific nested polymerase chain reaction (PCR) combined with DNA sequencing assay. As a result, Candidatus Rickettsia jingxinensis (24.61%, 127/516), Anaplasma marginale (13.18%, 68/516), Coxiella burnetii (3.10%, 16/516), and Coxiella-like endosymbiont (CLE) (8.33%, 43/516) were detected. The dual coinfection with Ca. R. jingxinensis and A. marginale and the triple coinfection with Ca. R. jingxinensis, A. marginale, and CLE were most frequent and detected in 3.68% (19/516) and 3.10% (16/516) of these ticks, respectively. The results provide insight into the diversity of TBPs and their coinfections in Rh. microplus in Yunnan province of China, reporting for the first time that C. burnetii had been found in Rh. microplus in China. Multilocus variable number tandem repeat analysis with 6 loci (MLVA-6) discriminated the C. burnetii detected in Rh. microplus in Yunnan into MLVA genotype 1, which is closely related to previously described genotypes found primarily in tick and human samples from different regions of the globe, indicating a potential public health threat posed by C. burnetii in Rh. microplus in Yunnan.

Study on candidate genes for milk production traits of Red Chittagong Cattle

The study was carried out on Stearoyl-CoA desaturase (SCD,) diacylglycerolacyltransferase-1 (DGAT1) and ATP-binding cassette G2 (ABCG2) genes which are responsible for variation in milk production traits (milk yield, fat yield, protein yield, and SNF yield) in cattle. These genes were used as candidate genes in Red Chittagong Cattle (RCC) breed of Bangladesh Livestock Research Institute (BLRI) herd for detection of single nucleotide polymorphisms (SNPs) causing variation in milk production traits. Focusing on the effects of SNPs on milk production traits, phenotypic variation within RCC breed was identified and categorized based on milk production traits. Average lactation yield varied from 527 to 1436 kg (n=29) per lactation. About 18% of lactating cows showed an average of >1000 kg per lactation. Average fat percent ranged from 4.71 to 6.25 (n=15). Eighteen (18) set of primers were designed to amplify targeted regions of SCD, DGAT1 and ABCG2 genes, where 8 set from DGAT1, 6 set from SCD and 4 set from ABCG2 gene. Pooled DNA from 50 RCC cows and 5 RCC bulls were used in sequencing. In sequence analysis, the SCD, DGAT1 and ABCG2 alleles found fixed in RCC. This study suggests an evidence that RCC breed has fixed alleles with respect to SCD, DGAT1 and ABCG2 genes reported to be responsible for higher milk fat yield, higher fat and protein percent. Bang. J. Livs. Res. Vol. 26 (1&2), 2019: P. 1-7

Genomic Variant Analyses in Pyrethroid Resistant and Susceptible Malaria Vector, Anopheles sinensis

Anopheles sinensis is a major malaria vector in Southeast Asia. Resistance to pyrethroid insecticides in this species has impeded malaria control in the region. Previous studies found that An. sinensis populations from Yunnan Province, China were highly resistant to deltamethrin and did not carry mutations in the voltage-gated sodium channel gene that cause knockdown resistance. In this study, we tested the hypothesis that other genomic variants are associated with the resistance phenotype. Using paired-end whole genome sequencing (DNA-seq), we generated 108 Gb of DNA sequence from deltamethrin -resistant and -susceptible mosquito pools with an average coverage of 83.3× depth. Using a stringent filtering method, we identified a total of 916,926 single nucleotide variants (SNVs), including 32,240 non-synonymous mutations. A total of 958 SNVs differed significantly in allele frequency between deltamethrin -resistant and -susceptible mosquitoes. Of these, 43 SNVs were present within 37 genes that code for immunity, detoxification, cuticular, and odorant proteins. A subset of 12 SNVs were randomly selected for genotyping of individual mosquitoes by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and showed consistent allele frequencies with the pooled DNA-seq derived allele frequencies. In addition, copy number variations (CNVs) were detected in 56 genes, including 33 that contained amplification alleles and 23 that contained deletion alleles in resistant mosquitoes compared to susceptible mosquitoes. The genomic variants described here provide a useful resource for future studies on the genetic mechanism of insecticide resistance in this important malaria vector species.

High standing genetic variation in an invasive plant allows immediate evolutionary response to climate warming

Predicting plant distributions under climate change is constrained by our limited understanding of potential rapid adaptive evolution. In an experimental evolution study with the invasive common ragweed, we subjected replicated populations of the same initial genetic composition to simulated climate warming. Pooled DNA sequencing of parental and offspring populations showed that warming populations experienced a greater loss of genetic diversity, and greater genetic divergence from their parents, than control populations. In a common environment, offspring from warming populations showed more convergent phenotypes in seven out of nine plant traits, with later flowering and larger biomass, than plants from control populations. For both traits, we also found a significant higher ratio of phenotypic to genetic differentiation across generations for warming than for control populations, indicating stronger selection under warming conditions. Our findings demonstrate that ragweed populations can rapidly evolve in response to climate change within a single generation.

The Application of UTY and SRY Molecular Markers for Determination of Unknown Sex Samples in Bali Cattle

An investigation involving large number of animals is often resulting incomplete or in accurate information such as animal parentage, or misidentify on sex due to unlabeled sex samples. A PCR method by applying Y chromosome markers (UTY and SRY) facilitates in determination of unknown sex problem. This study was intended to determine sex from unlabelled sex of blood samples by applying PCR method using a pooled-DNA template. Twenty five of unknown sex blood samples from Nusa Penida, Bali were used in this study. The samples were plotted into 5 pooled-DNA whith each pool DNA consisted of 5 individuals DNA. Two pairs of sex primers, UTY (58oC) and SRY (60oC) with 35 cycles were applied to amplify the samples. The result showed there was only one pooled-DNA (P4) amplified by UTY (484bp). Whereas re-PCR of the positive pooled-DNA (P4) using SRY primer, only one out of 25 samples determined as male Bali cattle (325bp). This finding suggests that UTY and SRY primers are suitable for sex determination and the pooled-DNA could be used as an efficient PCR method both in consumables and PCR process for sex determination. Keywords: Determination, sex, unknown sample, pooled DNA, Bali cattle.

Variations in terrestrial arthropod DNA metabarcoding methods recovers robust beta diversity but variable richness and site indicators

Terrestrial arthropod fauna have been suggested as a key indicator of ecological integrity in forest systems. Because phenotypic identification is expert-limited, a shift towards DNA metabarcoding could improve scalability and democratize the use of forest floor arthropods for biomonitoring applications. The objective of this study was to establish the level of field sampling and DNA extraction replication needed for arthropod biodiversity assessments from soil. Processing 15 individually collected soil samples recovered significantly higher median richness (488–614 sequence variants) than pooling the same number of samples (165–191 sequence variants) prior to DNA extraction, and we found no significant richness differences when using 1 or 3 pooled DNA extractions. Beta diversity was robust to changes in methodological regimes. Though our ability to identify taxa to species rank was limited, we were able to use arthropod COI metabarcodes from forest soil to assess richness, distinguish among sites, and recover site indicators based on unnamed exact sequence variants. Our results highlight the need to continue DNA barcoding local taxa during COI metabarcoding studies to help build reference databases. All together, these sampling considerations support the use of soil arthropod COI metabarcoding as a scalable method for biomonitoring.