Genomic DNA Pools Research Articles

Effect of SNPs in the Promoter Region on the Expression of Cytochrome P450 2E1 (CYP2E1) in Pig Liver.

Boar taint, an unfavorable odor in the meat of intact male pigs, is caused primarily by the accumulation of two compounds: androstenone and skatole. This multifactorial trait is regulated by numerous dietary, management and genetic factors. At the mechanistic level, there are many genes known to be involved in boar taint metabolism. Cytochrome P450 2E1 (CYP2E1) impacts boar taint through the phase I metabolism of skatole. The aim of this study was to identify single-nucleotide polymorphisms (SNPs) within the CYP2E1 gene promoter and explore their relationship with the expression of CYP2E1 mRNA and protein. Sequencing of the promoter region using pools of genomic DNA identified seven promoter region SNPs at -159, -586, -1693, -1806, -2322, -2369 and -2514 bp upstream of the ATG start site. Genomic DNA was obtained from 65 boars from the three major swine breeds: Duroc, Landrace and Yorkshire, and individual animals were genotyped for the identified SNPs. RNA was isolated from liver tissue and quantitative PCR was performed to measure CYP2E1 gene expression, while levels of CYP2E1 protein in liver were measured by Western blotting. Significant within-breed variation in CYP2E1 protein and mRNA expression was observed, indicating significant differences in gene expression among individuals. However, levels of CYP2E1 mRNA and protein were not significantly correlated. Two SNPs within the promoter were significantly associated with CYP2E1 mRNA expression, but not with protein expression. This study provides evidence of additional mutations affecting the gene expression of CYP2E1 and suggests that factors that affect the differences in translation of CYP2E1 mRNA may also be important in affecting skatole metabolism.

Method for detection of pathogenic bacteria from indoor air microbiome samples using high-throughput amplicon sequencing

The exposure of the air microbiome in indoor air posed a detrimental health effect to the building occupants compared to the outdoor air. Indoor air in hospitals has been identified as a reservoir for various pathogenic microbes. The conventional culture-dependent method has been widely used to access the microbial community in the air. However, it has limited capability in enumerating the complex air microbiome communities, as some of the air microbiomes are uncultivable, slow-growers, and require specific media for cultivation. Here, we utilized a culture-independent method via amplicon sequencing to target the V3 region of 16S rRNA from the pool of total genomic DNA extracted from the dust samples taken from hospital interiors. This method will help occupational health practitioners, researchers, and health authorities to efficiently and comprehensively monitor the presence of harmful air microbiome thus take appropriate action in controlling and minimizing the health risks to the hospital occupants. Key features;•Culture-independent methods offer fast, comprehensive, and unbias profiles of pathogenic and non-pathogenic bacteria from the air microbiomes.•Unlike the culture-dependent method, amplicon sequencing allows bacteria identification to the lowest taxonomy levels.

Gonococcal resistance to zoliflodacin could emerge via transformation from commensal Neisseria species. An in-vitro transformation study

One of the most promising new treatments for gonorrhoea currently in phase 3 clinical trials is zoliflodacin. Studies have found very little resistance to zoliflodacin in currently circulating N. gonorrhoeae strains, and in-vitro experiments demonstrated that it is difficult to induce resistance. However, zoliflodacin resistance may emerge in commensal Neisseria spp., which could then be transferred to N. gonorrhoeae via transformation. In this study, we investigated this commensal-resistance-pathway hypothesis for zoliflodacin. To induce zoliflodacin resistance, ten wild-type susceptible isolates belonging to 5 Neisseria species were serially passaged for up to 48 h on gonococcal agar plates containing increasing zoliflodacin concentrations. Within 7 to 10 days, all strains except N. lactamica, exhibited MICs of ≥ 4 µg/mL, resulting in MIC increase ranging from 8- to 64-fold. The last passaged strains and their baseline were sequenced. We detected mutations previously reported to cause zoliflodacin resistance in GyrB (D429N and S467N), novel mutations in the quinolone resistance determining region (QRDR) (M464R and T472P) and mutations outside the QRDR at amino acid positions 28 and 29 associated with low level resistance (MIC 2 µg/mL). Genomic DNA from the laboratory evolved zoliflodacin-resistant strains was transformed into the respective baseline wild-type strain, resulting in MICs of ≥ 8 µg/mL in most cases. WGS of transformants with decreased zoliflodacin susceptibility revealed presence of the same zoliflodacin resistance determinants as observed in the donor strains. Two inter-species transformation experiments were conducted to investigate whether zoliflodacin resistance determinants of commensal Neisseria spp. could be acquired by N. gonorrhoeae. N. gonorrhoeae strain WHO P was exposed to (i) pooled genomic DNA from the two resistant N. mucosa strains and (ii) a gyrB amplicon of the resistant N. subflava strain 45/1_8. Transformants of both experiments exhibited an MIC of 2 µg/mL and whole genome analysis revealed uptake of the mutations detected in the donor strains. This is the first in-vitro study to report that zoliflodacin resistance can be induced in commensal Neisseria spp. and subsequently transformed into N. gonorrhoeae.

Brief restriction endonuclease digestion of genomic DNA improves PCR amplifiability and reproducibility of SSR loci: Augmenting exploration of hidden genetic variability in an endemic carp of South-East Asia

Majority of the microsatellite primers developed through painstaking, resource intensive research often fail to amplify due to secondary structures in the repeat rich target. In the present work, we describe the use of restriction endonuclease digestion of genomic DNA prior to PCR to counter this issue. The methodology, Restriction Endonuclease Digestion of genomic DNA to improve PCR Amplifiability and Repeatability of SSR loci (REDDAR-SSR), was tested on 40 species-specific Simple Sequence Repeat (SSR) loci in Osteobrama belangeri (F: Cyprinidae). Undigested and HpaII digested pooled genomic DNA samples were used as templates. Two primer concentrations (10 pM and 25 pM) and two annealing temperatures (56 °C uniformly and individual primer-specific annealing temperatures) were tested on both template types in sets of three, repeated thrice (360 reactions/condition). The digested templates exhibited significantly higher (P < 0.05) amplifiability (93.9 % against 24.16 % of undigested counterpart) as well as repeatability (94.47 % against 21.56 % of undigested counterpart). Primer concentration had no significant effect on outcomes (P > 0.05). The number of successfully amplified loci in our method was 38 compared to 18 by the conventional protocol. All 38 loci could also be reliably amplified in individual gDNA using the developed protocol out of which 9 loci were observed to be polymorphic. Notably, 4 of the polymorohic loci could be amplified only through REDDAR-SSR. Our protocol can potentially reduce expenses; resources & time vis-a-vis enhance the reach of SSR markers for genotyping species of commercial or conservational significance.

Abstract 1138: Detection of genetically engineered iPSC-derived natural killer cells in blood and tissue

Abstract Immune cell therapies derived from induced pluripotent stem cells (iPSC) provide a novel opportunity for the treatment of multiple cancer types. Assessment of the persistence and biodistribution of these product candidates requires specific and sensitive methods to detect engineered cells in both liquid and solid biopsies. Here, we present the development and validation of two complimentary nucleic acid-based detection assays for iPSC-derived natural killer (iNK) cell product candidates containing Fate’s proprietary high-affinity, non-cleavable CD16 transgene (hnCD16). The first assay is a droplet digital PCR (ddPCR) method to detect and quantify hnCD16 transgene copies present in a pool of genomic DNA (gDNA). The primers and probe were designed to recognize the optimized codons of hnCD16. Assay linearity and accuracy were assessed through titration studies using 0.024 to 1 ng of hnCD16-containing DNA spiked into different amounts of hnCD16-negative gDNA. Precision was determined through multiple assay runs by different operators on two instruments. The second assay is an in situ hybridization based method utilizing RNAscope࣪ technology to detect cells expressing hnCD16 in fixed tissue. Probes targeting hnCD16 were used to optimize signal specificity. Cells expressing hnCD16 and tissues from in vivo studies treated with iNK products served as positive controls. For the ddPCR assay, absolute limit of detection (aLoD) was determined to be 4.9 copies of hnCD16 per 20 µL reaction, regardless of total genomic mass input. Absolute limit of quantification (aLoQ) was 12 copies per 20 µL reaction with a %CV ≤30. Relative limit of quantification (rLoQ), assessing transgene to total DNA ratio, is affected by the background gDNA input and is less sensitive with lower input mass. rLoQ for total mass of 70 - 250 ng was 97 - 22 copies/µg gDNA (0.064% - 0.015%) with a %CV ≤30. The sensitivity of this input range allows evaluation of clinical samples with low cellularity. While ddPCR provides robust quantification of the hnCD16 transcript, the RNAscope࣪ assay informs localization of the iNK product. Specificity of the probe was established by confirming its lack of affinity for endogenous CD16 using a variety of human normal and tumor tissues and by staining hnCD16-positive fixed cell pellets and tissues from in vivo studies. In cell pellets, positive RNAscope࣪ signal correlated with the known ratio of transgene positive cells. In murine tissues previously confirmed to contain iNK cell product, the RNAscope࣪ positive staining correlated with NKG2A immunohistochemistry staining, confirming the presence of product NK cells. The combined use of both the ddPCR and RNAscope࣪ assays targeted to hnCD16 allows for detection and quantification of transgene-bearing iNK cells in a wide variety of patient samples including tumor biopsies. Both assays are being utilized for cell detection and quantification in our ongoing clinical trials. Citation Format: Cara E. Bickers, Judy L. Martin, Steven Castro, Jason Zhang, Thomas Dailey, Eric Sung, Suzanna Gasparian, Jason O'Rourke, Moyar Ge, Tom T. Lee, Janel Huffman, Jode Goodridge, Ryan Bjordahl, Bahram Valamehr, Peter M. Szabo, Lilly Wong, Sarah Cooley. Detection of genetically engineered iPSC-derived natural killer cells in blood and tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1138.

Sex locus and sex markers identification using whole genome pool-sequencing approach in the largemouth bass (Micropterus Salmoides L.)

The mechanism of sex determination in teleost evolves in an amazing speed contrary to mammals and birds. Different populations of the same species may use different sex determination systems. Largemouth bass is an aquaculture importance and recreational fish, which belongs to the family of Centrarchidae, genus Micropterus. However, molecular basis of genetic sex determination currently remains unclear. We applied a whole genome pool-sequencing approach to better characterize sex determination in this fish. Our results confirmed that sex determination system in largemouth bass is male heterogametic (XX/XY). In this study, males and females genomic DNA pools were sequenced using a whole genomic sequencing approach. Using genome comparative analysis, we identified a large sex differentiated region (~ 1.7 Mb) at the beginning of chromosome 7 on largemouth bass. This non-recombination region contained enriched male-biased fragments and high-density of male-biased genetic polymorphisms. Meanwhile, sequence synteny analysis between/within sex chromosomes showed that many chromosomal rearrangement events have taken place in this region. Based on this genetic information, we successfully developed male specific genetic markers. Additionally, 96 genes were located on that sex differentiated region, whereas “usual suspects” sex determining gene didn't exist in this region. It is implied, in largemouth bass, a new gene has evolved as the master sex determining gene. Our results exhibited that largemouth bass has a large sex locus on Chromosome 7, which is likely the largemouth bass sex chromosome. The presence of numerous chromosomal rearrangements and the absence of “usual suspects” sex determining gene in this sex differentiated region indicate recombination is suppressed on that region and a new master sex determining gene may be co-opted in largemouth bass. Besides, we also developed sex-linked genetic markers. Our study will facilitate future researches on sex determination and aquaculture applications in largemouth bass.

Whole Exome-Sequencing of Pooled Genomic DNA Samples to Detect Quantitative Trait Loci in Esotropia and Exotropia of Strabismus in Japanese.

Background: Esotropia and exotropia are two major phenotypes of comitant strabismus. It remains controversial whether esotropia and exotropia would share common genetic backgrounds. In this study, we used a quantitative trait locus (QTL)-sequencing pipeline for diploid plants to screen for susceptibility loci of strabismus in whole exome sequencing of pooled genomic DNAs of individuals. Methods: Pooled genomic DNA (2.5 ng each) of 20 individuals in three groups, Japanese patients with esotropia and exotropia, and normal members in the families, was sequenced twice after exome capture, and the first and second sets of data in each group were combined to increase the read depth. The SNP index, as the ratio of variant genotype reads to all reads, and Δ(SNP index) values, as the difference of SNP index between two groups, were calculated by sliding window analysis with a 4 Mb window size and 10 kb slide size. The rows of 200 “N”s were inserted as a putative 200-b spacer between every adjoining locus to depict Δ(SNP index) plots on each chromosome. SNP positions with depth < 20 as well as SNP positions with SNP index of <0.3 were excluded. Results: After the exclusion of SNPs, 12,242 SNPs in esotropia/normal group and 12,108 SNPs in exotropia/normal group remained. The patterns of the Δ(SNP index) plots on each chromosome appeared different between esotropia/normal group and exotropia/normal group. When the consecutive groups of SNPs on each chromosome were set at three patterns: SNPs in each cytogenetic band, 50 consecutive sliding SNPs, and SNPs in 4 Mb window size with 10 kb slide size, p values (Wilcoxon signed rank test) and Q values (false discovery rate) in a few loci as Manhattan plots showed significant differences in comparison between the Δ(SNP index) in the esotropia/normal group and exotropia/normal group. Conclusions: The pooled DNA sequencing and QTL mapping approach for plants could provide overview of genetic background on each chromosome and would suggest different genetic backgrounds for two major phenotypes of comitant strabismus, esotropia and exotropia.

Fluorescence lifetime imaging for studying DNA compaction and gene activities

Optical imaging is a most useful and widespread technique for the investigation of the structure and function of the cellular genomes. However, an analysis of immensely convoluted and irregularly compacted DNA polymer is highly challenging even by modern super-resolution microscopy approaches. Here we propose fluorescence lifetime imaging (FLIM) for the advancement of studies of genomic structure including DNA compaction, replication as well as monitoring of gene expression. The proposed FLIM assay employs two independent mechanisms for DNA compaction sensing. One mechanism relies on the inverse quadratic relation between the fluorescence lifetimes of fluorescence probes incorporated into DNA and their local refractive index, variable due to DNA compaction density. Another mechanism is based on the Förster resonance energy transfer (FRET) process between the donor and the acceptor fluorophores, both incorporated into DNA. Both these proposed mechanisms were validated in cultured cells. The obtained data unravel a significant difference in compaction of the gene-rich and gene-poor pools of genomic DNA. We show that the gene-rich DNA is loosely compacted compared to the dense DNA domains devoid of active genes.

Genomic and cDNA selection-amplification identifies transcriptome-wide binding sites for the Drosophila protein sex-lethal.

BackgroundDownstream targets for a large number of RNA-binding proteins remain to be identified. The Drosophila master sex-switch protein Sex-lethal (SXL) is an RNA-binding protein that controls splicing, polyadenylation, or translation of certain mRNAs to mediate female-specific sexual differentiation. Whereas some targets of SXL are known, previous studies indicate that additional targets of SXL have escaped genetic screens.Methodology/Principal findingsHere, we have used an alternative molecular approach of GEnomic Selective Enrichment of Ligands by Exponential enrichment (GESELEX) using both the genomic DNA and cDNA pools from several Drosophila developmental stages to identify new potential targets of SXL. Our systematic analysis provides a comprehensive view of the Drosophila transcriptome for potential SXL-binding sites.Conclusion/SignificanceWe have successfully identified new SXL-binding sites in the Drosophila transcriptome. We discuss the significance of our analysis and that the newly identified binding sites and sequences could serve as a useful resource for the research community. This approach should also be applicable to other RNA-binding proteins for which downstream targets are unknown.

Identification of two QTLs associated with high fruit acidity in apple using pooled genome sequencing analysis

Acidity is a critical component determining apple fruit quality. Previous studies reported two major acidity quantitative trait loci (QTLs) on linkage groups (LGs) 16 (Ma) and 8 (Ma3), respectively, and their homozygous genotypes mama and ma3ma3 usually confer low titratable acidity (TA) (<3.0 mg ml−1) to apple fruit. However, apples of genotypes Ma- (MaMa and Mama) or Ma3- (Ma3Ma3 and Ma3ma3) frequently show an acidity range spanning both regular (TA 3.0–10.0 mg ml−1) and high (TA > 10 mg ml−1) acidity levels. To date, the genetic control for high-acidity apples remains essentially unknown. In order to map QTLs associated with high acidity, two genomic DNA pools, one for high acidity and the other for regular acidity, were created in an interspecific F1 population Royal Gala (Malus domestica) × PI 613988 (M. sieversii) of 191 fruit-bearing progenies. By Illumina paired-end sequencing of the high and regular acidity pools, 1,261,640 single-nucleotide variants (SNVs) commonly present in both pools were detected. Using allele frequency directional difference and density (AFDDD) mapping approach, one region on chromosome 4 and another on chromosome 6 were identified to be putatively associated with high acidity, and were named Ma6 and Ma4, respectively. Trait association analysis of DNA markers independently developed from the Ma6 and Ma4 regions confirmed the mapping of Ma6 and Ma4. In the background of MaMa, 20.6% of acidity variation could be explained by Ma6, 28.5% by Ma4, and 50.7% by the combination of both. The effects of Ma6 and Ma4 in the background of Mama were also significant, but lower. These findings provide important genetic insight into high acidity in apple.

Culicidae-centric metabarcoding through targeted use of D2 ribosomal DNA primers.

A practical limitation to many metabarcoding initiatives is that sampling methods tend to collect many non-target taxa, which become “amplicon noise” that can saturate Next Generation Sequencing results and lead to both financial and resource inefficiencies. An available molecular tool that can significantly decrease these non-target amplicons and decrease the need for pre-DNA-extraction sorting of bycatch is the design of PCR primers tailored to the taxa under investigation. We assessed whether the D2 extension segment of the 28S ribosomal operon can limit this shortcoming within the context of mosquito (Culicidae) monitoring. We designed PCR primers that are fully conserved across mosquitos and exclude from amplification most other taxa likely to be collected with current sampling apparatuses. We show that, given enough sequencing depth, D2 is an effective marker for the detection of mosquito sequences within mock genomic DNA pools. As few as 3,050 quality-filtered Illumina reads were able to recover all 17 species in a bulk pool containing as little as 0.2% of constituent DNA from single taxa. We also mixed these mosquito DNA pools with high concentrations of non-Culicidae bycatch DNA and show that the component mosquito species are generally still recoverable and faithful to their original relative frequencies. Finally, we show that there is little loss of fidelity in abundance parameters when pools from degraded DNA samples were sequenced using the D2 primers.

Association of TLR gene variants in a Czech Red Pied cattle population with reproductive traits

The bovine genes TLR1, TLR2 and TLR6, which encode Toll-like receptors, key components of the innate immune system, were screened for polymorphisms in Czech Red Pied (Czech Simmental) cattle, and the different variants present in the population were tested for association with reproductive and fitness traits. Diversity was investigated in a group of 164 bulls using hybrid resequencing of pooled amplicons with PacBio technology and of pooled genomic DNA using HiSeq X-Ten technology. The validated single nucleotide polymorphisms (SNPs) were genotyped in individual animals using the primer extension technique. The association of genotypic classes of 16 polymorphisms with six phenotypic traits were estimated with one-way analysis of variance (ANOVA) and with restricted maximum likelihood (REML) algorithm. The evaluated traits included the incidence of cystic ovaries, index of early reproductive disorders, paternal and maternal indicators of calving ease, production longevity and calf vitality index. The estimated breeding values were used for combined trait quantification. Early traits, namely, cystic ovaries and early reproductive disorders, were not associated with any of the tested polymorphisms according to the general ANOVA test. By contrast, five variants of all three genes were associated with calving ease, both paternal and maternal. The production longevity correlated with two variants of TLR1 and the calf vitality index correlated with the 1044 T > C (rs68268249) polymorphism in TLR2. The false discovery rate (FDR) according to Benjamini-Hochberg was favourable for the calving ease trait (0.221) and maternal calving ease (0.214), which allows to consider the observed associations real, regardless of the error arising from the multiple comparisons. These results were supported by REML only partially, probably in view of the additivity assumption. Two mechanisms of action on calving are conceivable, either via infection resistance or via the involvement of TLR2 in signalling in the myometrium. The known formation of heterodimers by the TLR1, -2 and -6 products might be responsible for the shared pattern of action in these genes. The association of the calf vitality index with TLR2 variation might reflect the increased role of infections in calves compared to adult animals.

Genome-wide re-sequencing and transcriptome analysis reveal candidate genes associated with the pendulous comb phenotype in domestic chickens.

To determine the causative variations associated with two chicken comb phenotypes, pendulous comb (PC) or upright comb (UC), two pooled genomic DNA samples from PC and UC chickens were re-sequenced by Next-Generation Sequencer, and genome-wide Single nucleotide polymorphisms (SNPs) were detected. Using three selective sweep approaches, FST , θπ, and Tajima's D, with top 5% window values serving as the threshold, a total of 84 positively selective genes (PSGs) were identified. There were no SNPs in exons of the PSGs with significant differences in allele frequencies between the two comb phenotype groups. Then, 515 differentially expressed genes (DEGs) between the PC and UC were identified by RNA-seq. Three genes including CD36 (CD36 molecule), ADAMTSL3 (ADAMTS-like 3), and AOX1 (aldehyde oxidases 1) are overlapped between PSGs and DEGs. After genotyping seven candidate SNPs in the regulatory regions of the three overlapping genes in 120 chickens from two other breeds, two variants (rs14607046 and rs731818051) in the regulatory regions of AOX1 and ADAMTSL3 were found to have significant differences in allele frequency between the PC and UC, suggesting that the two variants may be causative mutations for PC. Overall, our study shed light on the genetic basis underlying the PC phenotype in chickens.

A High-Content Screen Identifies MicroRNAs That Regulate Liver Repopulation After Injury in Mice

Liver regeneration is impaired in mice with hepatocyte-specific deficiencies in microRNA (miRNA) processing, but it is not clear which miRNAs regulate this process. We developed a high-throughput screen to identify miRNAs that regulate hepatocyte repopulation after toxic liver injury using fumarylacetoacetate hydrolase-deficient mice. We constructed plasmid pools encoding more than 30,000 tough decoy miRNA inhibitors (hairpin nucleic acids designed to specifically inhibit interactions between miRNAs and their targets) to target hepatocyte miRNAs in a pairwise manner. The plasmid libraries were delivered to hepatocytes in fumarylacetoacetate hydrolase-deficient mice at the time of liver injury via hydrodynamic tail-vein injection. Integrated transgene-containing transposons were quantified after liver repopulation via high-throughput sequencing. Changes in polysome-bound transcripts after miRNA inhibition were determined using translating ribosome affinity purification followed by high-throughput sequencing. Analyses of tough decoy abundance in hepatocyte genomic DNA and input plasmid pools identified several thousand miRNA inhibitors that were significantly depleted or increased after repopulation. We classified a subset of miRNA binding sites as those that have strong effects on liver repopulation, implicating the targeted hepatocyte miRNAs as regulators of this process. We then generated a high-content map of pairwise interactions between 171 miRNA-binding sites and identified synergistic and redundant effects. We developed a screen to identify miRNAs that regulate liver repopulation after injury in live mice.

1550P - Preferential expression of the affected MET allele in lung carcinomas with heterozygous MET exon 14 skipping mutations: Implications for clinical testing

BackgroundLung carcinomas (LCs) carrying MET exon 14 skipping (exon 14Δ) mutations are responsive to a number of tyrosine kinase inhibitors. There is a need to incorporate MET analysis in the diagnostic pipeline for LC patients. MethodsNucleic acids were extracted from formalin-fixed paraffin-embedded archival LC samples and subjected to cDNA synthesis. This pool of genomic DNA and cDNA was sequentially tested for EGFR, ALK and MET mutations by PCR-based assays. ResultsAllele-specific PCR detected MET exon 14 skipping in 35/1415 (2.5%) EGFR mutation–negative LCs. There was a highly pronounced association with the elderly age of the patients (median age 69 years as compared to 62 years in EGFR/ALK/MET mutation-negative cases; p=1.821e-06). 34/35 (97%) LCs with MET exon 14 skipping mutations showed preferential expression of the affected MET allele, while the wild-type transcript was almost undetectable in these tumors. In addition, we identified a subset of MET wild-type LCs, which produced normal MET transcript but also expressed residual amounts of MET exon 14Δ RNA message, probably due to alternative splicing. ConclusionsThe mere detection of MET exon 14Δ signal by allele-specific PCR does not warrant the presence of corresponding clonal MET mutation. Comparison of expression of MET exon 14Δ and wild-type alleles is essential for reliable identification of tumors carrying drug-sensitizing MET lesions. Legal entity responsible for the studyThe authors. FundingRussian Science Foundation (grant 17-75-30027). DisclosureAll authors have declared no conflicts of interest.

Whole-Genome Resequencing Reveals Loci Associated With Thoracic Vertebrae Number in Sheep.

The number of vertebrae, especially thoracic vertebrae, is an important economic trait that may influence carcass length and meat production in animals. However, the genetic basis of vertebrae number in sheep is still poorly understood. To detect the candidate genes, 400 increased number of thoracic vertebrae (T14L6) and 200 normal (T13L6) Kazakh sheep were collected. We generated and sequenced 60 pools of genomic DNA (each pool prepared by mixing genomic DNA from 10 sheep with the same thoracic traits), with an average depth of coverage of 25.65×. We identified a total of 42,075,402 SNPs and 11 putatively selected genomic regions, including the VRTN gene and the HoxA gene family that regulate vertebral development. The most prominent areas of selective elimination were located in a region of chromosome 7, including VRTN, which regulates spinal development and morphology. Further investigation indicated that the expression level of the VRTN gene during fetal development was significantly higher in sheep with more thoracic vertebrae than in those with a normal number of thoracic vertebrae. A genome-wide comparison between sheep with increased and normal numbers of thoracic vertebrae showed that the VRTN gene is the major selection locus for the number of thoracic vertebrae in sheep and has the potential to be utilized in sheep breeding in the future.

Complete genome sequences of pooled genomic DNA from 10 marine bacteria using PacBio long-read sequencing

BackgroundHigh-quality, completed genomes are important to understand the functions of marine bacteria. PacBio sequencing technology provides a powerful way to obtain high-quality completed genomes. However individual library production is currently still costly, limiting the utility of the PacBio system for high-throughput genomics. Here we investigate how to generate high-quality genomes from pooled marine bacterial genomes. ResultsPooled genomic DNA from 10 marine bacteria were subjected to a single library production and sequenced with eight SMRT cells on the PacBio RS II sequencing platform. In total, 7.35 Gbp of long-read data was generated, which is equivalent to an approximate 168× average coverage for the input genomes. Genome assembly showed that eight genomes with average nucleotide identities (ANI) lower than 91.4% can be assembled with high-quality and completion using standard assembly algorithms (e.g. HGAP or Canu). A reference-based reads phasing step was developed and incorporated to assemble the complete genomes of the remaining two marine bacteria that had an ANI > 97% and whose initial assemblies were highly fragmented. ConclusionsTen complete high-quality genomes of marine bacteria were generated. The findings and developments made here, including the reference-based read phasing approach for the assembly of highly similar genomes, can be used in the future to design strategies to sequence pooled genomes using long-read sequencing.

Genetic diversity of avocado (Persea americana Mill.) germplasm using pooled sequencing

BackgroundDiscovering a genome-wide set of avocado (Persea americana Mill.) single nucleotide polymorphisms and characterizing the diversity of germplasm collection is a powerful tool for breeding. However, discovery is a costly process, due to loss of loci that are proven to be non-informative when genotyping the germplasm.ResultsOur study on a collection of 100 accessions comprised the three race types, Guatemalan, Mexican, and West Indian. To increase the chances of discovering polymorphic loci, three pools of genomic DNA, one from each race, were sequenced and the reads were aligned to a reference transcriptome. In total, 507,917 polymorphic loci were identified in the entire collection. Of these, 345,617 were observed in all three pools, 117,692 in two pools, 44,552 in one of the pools, and only 56 (0.0001%) were homozygous in the three pools but for different alleles. The polymorphic loci were validated using 192 randomly selected SNPs by genotyping the accessions within each pool. The sensitivity of polymorphic locus prediction ranged from 0.77 to 0.94. The correlation between the allele frequency estimated from the pooled sequences and actual allele frequency from genotype calling of individual accessions was r = 0.8. A subset of 109 SNPs were then used to evaluate the genetic relationships among avocado accessions and the genetic diversity of the collection. The three races were distinctly clustered by projecting the genetic variation on a PCA plot. As expected, by estimating the kinship coefficient for all the accessions, many of the cultivars from the California breeding program were closely related to each other, especially, the Hass-like ones. The green-skin avocados, e.g., ‘Bacon’, ‘Zutano’, ‘Ettinger’ and ‘Fuerte’ were also closely related to each other.ConclusionsA framework for SNP discovery and genetically characterizing of a breeder‘s accessions was described. Sequencing pools of gDNA is a cost-effective approach to create a genome-wide stock of polymorphic loci for a breeding program. Reassessing the botanical and the genetic knowledge about the germplasm accessions is valuable for future breeding. Kinship analysis may be used as a first step in finding a parental candidates in a parentage analyses.

Higher‐level phylogeny and evolutionary history of Pentatomomorpha (Hemiptera: Heteroptera) inferred from mitochondrial genome sequences

AbstractThe higher‐level phylogeny of Pentatomomorpha, the second largest infraorder of true bugs (Hemiptera: Heteroptera), which includes many important agriculture and forestry pests, has been debated for decades. To investigate the phylogeny and evolutionary history of Pentatomomorpha, we assembled new mitochondrial genomes for 46 species through next‐generation sequencing of pooled genomic DNA. Based on a much broader taxon sampling than available previously, Bayesian analyses using a site‐heterogeneous mixture model (CAT+GTR) resolved the higher‐level phylogeny of Pentatomomorpha as (Aradoidea + (Pentatomoidea + (Coreoidea + (Lygaeoidea + Pyrrhocoroidea)))). There was a transition from trnT/trnP to trnP/trnT in the common ancestor of Pyrrhocoroidea, which indicates that this gene rearrangement could be an autapomorphy for Pyrrhocoroidea. Divergence time analyses estimated that Pentatomomorpha originated c. 242 Ma in the Middle Triassic, and most of the recognized superfamilies originated during the Middle Jurassic to Early Cretaceous. The diversification of families within Pentatomomorpha largely coincided with the radiation of angiosperms during the Early Cretaceous.

Using Whole Genome Sequencing (WGS) of 3256 Patients with Hematologic Malignancies to Determine Genome Instability

Background:Genome instability is a hallmark of cancer. Mutations in DNA repair pathway genes are frequent in a number of solid tumors. Defects in DNA repair or damage response can weaken response to conventional chemotherapy and are frequently regarded as poor prognostic markers. However, a high tumor mutation burden (TMB, number of somatic mutations per mega base) was recently found to correlate with better response to immune checkpoint inhibitors e.g. in colon cancer. Patients with defects in the DNA mismatch repair (MMR) pathway in solid tumors are among the cases with the highest TMB. Hematological malignancies are generally expected on the lower end of the TMB spectrum. We used whole genome sequencing (WGS) for 3256 patients with hematological malignancies (lymphatic and myeloid) to determine factors of genetic instability across all entities.Aim:To determine the number of known mutations in genes from the DNA repair pathwayTo estimate TMB using WGS and identify cases with high TMB in hematologic malignanciesMethods:We investigated a cohort of 3256 patients with hematological malignancies, who were analyzed according to WHO diagnostic gold standards for routine purposes (incl. 584 acute myeloid leukemia [AML] and 635 myelodysplastic syndromes [MDS] samples). We performed amplification-free library preparation and sequencing on HiseqX and NovaSeq 6000 with a median coverage of 106x. Mapping and variant calling was performed with standard pipelines via BaseSpace (all Illumina, San Diego, CA). A pool of gender-matched genomic DNA (Promega, Madison, WI) was used for a tumor-unmatched normal variant calling. (a) In detail we evaluated 180 genes involved in DNA repair. We filtered on (likely) pathogenic variants from ClinVar and for TP53 on protein-truncating variants and (likely, possibly) pathogenic variants from the IARC database. (b) For TBM calculation we determined protein-altering changes and then subtracted all gnomAD listed variants in order to eliminate most germline variants.Results:We found 479 of 3256 (15%) patients with at least one pathogenic variant according to current database annotations in DNA repair or damage response genes. Most pathogenic variants were found in TP53 (330/3256; 10%) and ATM (25/3256, 1%), however, this is probably the effect of the already available systematic database annotation for both genes. For routine diagnostic purposes TP53 mutation status had been analyzed for 1184 patients with Sanger sequencing (7%) or amplicon next-generation sequencing (93%). A 98% and 99% concordance of the pathogenic and non-pathogenic TP53 status was found in comparison to WGS. Mutations in genes from the DNA double-strand break repair (and/or homologous DNA pairing and strand exchange) pathway were found in 93 patients (3%). Pathogenic and potentially germline MMR gene mutations were found in only 3 patients (0.1%, 2 MLH1, 1 MSH6), which equals the expected frequency in the Western population (0.05-0.3%).Next, we calculated TMB. The average was 2.4 [range: 0.4-39.2]. Only samples above the 95th percentile were defined as “TMBhi” (TMB ≥5). TMB was lowest in chronic myeloid leukemia (CML) and essential thrombocythemia (ET) (< 2) and no ET or CML patient was found among the TMBhi. We then focused on MDS, which is our largest subcohort: 56 of 635 (9%) patients were in TMBhi. Furthermore, among MDS patients, a significantly higher TMB was found in MDS-EB-2 (average 3.3 vs. 2.3 for non EB-2, p<.001) and a significantly lower TMB in MDS with isolated del(5q) (1.7 vs. 2.6 in all other MDS, p<.001).There was no association of high TMB and annotated DNA repair gene mutations. The three MMR deficient patients had an average TMB (1.8-2.2) in the hematologic sample. However, TMB was positively correlated with age (Spearman's rank correlation, p<.001) and average TMB was lower in patients under 60 years (2.3 vs. 2.5, p=0.007, t-test).Conclusions:High TMB is rare in hematological neoplasms, and our data across all entities suggest that the acquisition of mutations over age should be a major contributing factor.While TP53 and a few other factors are well studied, using genome-wide datasets in the near future will allow to deeply understand the broad patterns or signatures of genome stability (incl. copy number or structural variants) and their prognostic or predictive value. DisclosuresBaer:MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Höllein:MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.