Indels In Sequences Research Articles

The incidence of donor white blood cell survival (transfusion-associated microchimerism) in Australian pediatric patients.

Donor leucocyte survival following red blood cell (RBC) transfusion, known as transfusion-associated microchimerism (TAM), can occur in some patients. In Australia, despite the introduction of leucocyte filtration (leucodepletion) during RBC manufacture, TAM has been detected in adult trauma patients. However, the incidence of TAM in Australian pediatric patients has not been analyzed. Patients aged 0-16 years were recruited across two cohorts. Retrospective participants had RBC transfusion between January 1, 2002 and November 15, 2017 and prospective participants received RBC transfusion between December 1, 2016 and November 25, 2020. Twelve bi-allelic insertion/deletion (InDel) polymorphisms were used to detect microchimerism amplification patterns using real-time PCR (RT-PCR) and droplet digital PCR (ddPCR). Of the retrospective cohort (n = 40), six patients showed amplification of InDel sequences indicating potential microchimerism. For three patients, minor InDel sequences were detected using RT-PCR only, two patients had minor InDel amplification using ddPCR only, and one patient had minor InDel amplification that was confirmed using both techniques. Amplification of minor sequences occurred in three patients who had received a bone marrow transplant in addition to RBC transfusion. In the prospective cohort (n = 25), no InDel amplification indicating potential microchimerism was detected using RT-PCR. Cell-based therapies had been administered in three patients where microchimerism amplification patterns were detected. Three patients have microchimerism that may be attributed to RBC transfusion. In prospective patients, who received leucodepleted and gamma-irradiated RBC units, no potential microchimerism amplification were detected. ddPCR may be a suitable technique for TAM analysis but requires further evaluation.

Identification of a major QTL region and candidate genes responsible for harvest date combining QTL mapping and QTL-seq approaches in walnut (Juglans regia L.)

Walnut (Juglans regia L.) takes place within the Juglandaceae family and is one of the most popular nut species. In walnut breeding, several limiting factors need to be revealed regarding the genetic mechanism underlying important agronomical traits. In the current report, the monitoring of the ‘Chandler x Kaplan-86’ F1 walnut population was carried out four successive years (2015, 2016, 2017, 2018) and the distribution of the plants for harvest date from four years was ranged from August 27th to October 22nd. Then, we dissected the genomic regions associated with harvest date using classical Quantitative Trait Loci (QTLs) mapping and a rapid and cost-effective QTL-seq approach in ‘Chandler x Kaplan-86’ F1 walnut population for the first time. In classical QTL mapping, a major QTL was recognized in LG4 corresponding to Chr1 in Chandler female map with 27.6 (%) phenotyping variance. In QTL-seq, we bulked the DNAs of the genotypes with extreme harvest date separately, the whole genome resequencing was performed for a parent Chandler and both bulks (early and late) to calculate Delta SNP indexes for each Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletion (InDels). Approximately 48.04 million paired reads for Chandler, 149.72, and 150.14 million paired reads were obtained from early and late harvest bulks, respectively. A total of 229,307 SNPs and 45,223 InDels were mined from the merged sequences data covered whole chromosomes. The responsible genomic fragments were identified in Chr2 and Chr11 in QTL-seq. The highest linked InDel locus detected in QTL-seq in Chr11 around 17 million base pairs distinguished the plants according to harvest date. InDel Sequence Characterized Amplified Region (SCAR) marker is a candidate for marker-assisted selection for future walnut breeding studies.

Development of a Gene-Based Soybean-Origin Discrimination Method Using Allele-Specific Polymerase Chain Reaction.

A low soybean self-sufficiency rate in South Korea has caused a high import dependence and considerable price variation between domestic and foreign soybeans, causing the false labeling of foreign soybeans as domestic. Conventional soybean origin discrimination methods prevent a single-grain analysis and rely on the presence or absence of several compounds or concentration differences. This limits the origin discrimination of mixed samples, demonstrating the need for a method that analyzes individual grains. Therefore, we developed a method for origin discrimination using genetic analysis. The whole-genome sequencing data of the Williams 82 reference cultivar and 15 soybean varieties cultivated in South Korea were analyzed to identify the dense variation blocks (dVBs) with a high single-nucleotide polymorphism density. The PCR primers were prepared and validated for the insertion-deletion (InDel) sequences of the dVBs to discriminate each soybean variety. Our method effectively discriminated domestic and foreign soybean varieties, eliminating their false labeling.

Exploring Genetic Markers: Mitochondrial DNA and Genomic Screening for Biodiversity and Production Traits in Donkeys.

Donkeys (Equus asinus) play a pivotal role as essential livestock in arid and semi-arid regions, serving various purposes such as transportation, agriculture, and milk production. Despite their significance, donkey breeding has often been overlooked in comparison to other livestock species, resulting in limited genetic improvement programs. Preserving donkey genetic resources within each country necessitates the establishment of breed conservation programs, focusing on managing genetic diversity among populations. In recent years, significant strides have been made in sequencing and analyzing complete mitochondrial DNA (mtDNA) molecules in donkeys. Notably, numerous studies have honed in on the mitochondrial D-loop region, renowned for its remarkable variability and higher substitution rate within the mtDNA genome, rendering it an effective genetic marker for assessing genetic diversity in donkeys. Furthermore, genetic markers at the RNA/DNA level have emerged as indispensable tools for enhancing production and reproduction traits in donkeys. Traditional animal breeding approaches based solely on phenotypic traits, such as milk yields, weight, and height, are influenced by both genetic and environmental factors. To overcome these challenges, genetic markers, such as polymorphisms, InDel, or entire gene sequences associated with desirable traits in animals, have achieved widespread usage in animal breeding practices. These markers have proven increasingly valuable for facilitating the selection of productive and reproductive traits in donkeys. This comprehensive review examines the cutting-edge research on mitochondrial DNA as a tool for assessing donkey biodiversity. Additionally, it highlights the role of genetic markers at the DNA/RNA level, enabling the informed selection of optimal production and reproductive traits in donkeys, thereby driving advancements in donkey genetic conservation and breeding programs.

Simplified CRISPR-Mediated DNA Editing in Multicellular Eukaryotes.

The CRISPR-Cas9 system is becoming an imperative tool to edit the genome of various organisms. The gene-editing study by the CRISPR-Cas9 system has revolutionized the diverse field of biomedical research, genome engineering, and gene therapy. CRISPR-Cas9 system has been modified to induce genome editing by small-guide RNAs, which function together with Cas9 nuclease for sequence-specific cleavage of target sequences. Here, we describe the simplified protocol of CRISPR-Cas9-mediated DNA editing in multicellular eukaryotes, including the construction of gRNA plasmids into vectors, screening of positive clones, transfections into 293FT cell line, and transduction into Jurkat cells. We also describe different bioinformatic tools to design suitable gRNAs with increased efficiency and decreased off-target events. Further, we describe the assessments of DNA editing by indel mutations and sequencing in transduced cells.

Mining candidate genes for rice cadmium accumulation in the shoot through a genome-wide association study and transcriptomic analysis.

High cadmium (Cd) accumulation in rice is a serious threat to human health. The genetic mechanism of Cd accumulation in rice is highly complicated. To identify the low Cd accumulation in rice germplasm, investigate the genetic mechanism underlying Cd accumulation, and mine the elite genes of significant importance for rice breeding of low Cd accumulation varieties, we performed a genome-wide association study (GWAS) for rice Cd concentration in the shoot. The rice accessions were 315 diverse indica rice accessions selected from the 1568 rice accessions with 700,000 SNPs. Within the high rate of linkage disequilibrium (LD) decay, eight QTLs related to rice Cd accumulation were identified. Transcriptomic analysis showed there were 799 differentially expressed genes (DEGs) in the root and 857 DEGs in the shoot, which are probably considered to be the cause of the significant difference in Cd accumulation between high and low Cd accumulation varieties. In qCd11-1, we detected a crucial candidate gene, LOC_Os11g11050, which encodes an initiation factor, expressed differently in the root between the high and low Cd accumulation varieties. Furthermore, under Cd treatment, the expression levels of LOC_Os11g11050 significantly decreased in both the high and low Cd accumulation varieties. Sequence comparison and qRT-PCR revealed that there were indel sequences and base substitutions in the promoter region of LOC_Os11g11050 correlated with the LOC_Os11g11050 expression level, as well as the phenotype of Cd concentration differences in shoot between the high and low Cd accumulation accessions. LOC_Os11g11050 might play important roles in Cd accumulation. The results of our study provide valuable resources for low Cd accumulation in indica varieties and the candidate functional gene, as well as molecular mechanisms for Cd accumulation in indica rice. The genetic architecture underlying Cd accumulation in indica can be used for further applying the low Cd gene existing in indica for decreasing Cd accumulation in rice.

Loss of connective tissue growth factor permits mesenchymal transition in ovarian cancer (260)

<b>Objectives:</b> Epithelial-mesenchymal transition (EMT) is a characteristic feature of ovarian cancer (OC) metastasis. Our study aimed to determine the role of Connective Tissue Growth Factor (CTGF) in EMT. <b>Methods:</b> R182 and R2615 are epithelial OC cell lines that are CK18+, b-catenin+, TWIST -, and vimentin+. Time-course experiments +/- Transforming Growth Factor-b (TGF-b) and a TGF-b inhibitor were performed to evaluate the timing of CTGF expression in EMT. R182 and R2615 CTGF knockouts (KO) were derived utilizing a Cas9/CRISPR-Cas9 lentivirus plasmid vector. Phenotypic characterization of wild types (WT) and CTGF KO cell lines were performed with the assays, including scratch wound, proliferation, and anoikis resistance assays. Scratch wound assays were treated with 50 ng/mL and 100 ng/mL concentrations of recombinant CTGF for 48 hours (hr). Cells were plated in triplicate, and confluence was measured. Proliferation was determined by cell count using Biotek Cytation over 72 hr. For anoikis resistance, cells were plated in triplicate in an ultra-low adhesive cell plate with cell growth media. Using Promega CellTiter assay, cell viability was quantified by absorbance at 490 nm at the following time points: 1hr, 24hr, 48hr, and 120hr. Western blots were performed to evaluate the expression of epithelial and mesenchymal markers. RNA sequence analysis was performed, and pathway analysis was evaluated using the iPathway guide. <b>Results:</b> TGF-b induced CTGF expression in both OC cell lines tested. CTGF was induced as early as 30 minutes post-treatment, continued to increase until 6hr, and expression was maintained until 24 hr. No CTGF expression was seen in the mesenchymal mR182 and mR2615 OC cell lines. CTGF KO was confirmed by western blot analysis and indel sequencing. CTGF KO had no effect on the expression of epithelial markers, b-catenin, CK18, and vimentin, but SNAIL, a mesenchymal marker, was induced. Loss of CTGF also had no effect on cell proliferation but promoted anoikis resistance. R182 CTGF KO cells displayed 80% viability when grown in detached conditions for 48hr compared to only 20% viability in WT cells. At 120hr, 30% of KO cells remain viable compared to <10% for WT cells. R2615 WT and KO cells demonstrated similar results. Finally, the scratch assay showed that CTGF KO cells decrease the time to confluence compared to WT. Administration of recombinant CTGF does not rescue the phenotype to WT levels.Fig. 1 <b>Conclusions:</b> CTGF is expressed early in epithelial OC cells during EMT and is regulated by TGF-b. Our data suggest that CTGF permits maintenance of an epithelial phenotype through inhibition of SNAIL expression, and loss of CTGF should be evaluated as a poor prognosis indicator in OC.

Targeting double-strand break indel byproducts with secondary guide RNAs improves Cas9 HDR-mediated genome editing efficiencies

Programmable double-strand DNA breaks (DSBs) can be harnessed for precision genome editing through manipulation of the homology-directed repair (HDR) pathway. However, end-joining repair pathways often outcompete HDR and introduce insertions and deletions of bases (indels) at the DSB site, decreasing precision outcomes. It has been shown that indel sequences for a given DSB site are reproducible and can even be predicted. Here, we report a general strategy (the “double tap” method) to improve HDR-mediated precision genome editing efficiencies that takes advantage of the reproducible nature of indel sequences. The method simply involves the use of multiple gRNAs: a primary gRNA that targets the wild-type genomic sequence, and one or more secondary gRNAs that target the most common indel sequence(s), which in effect provides a “second chance” at HDR-mediated editing. This proof-of-principle study presents the double tap method as a simple yet effective option for enhancing precision editing in mammalian cells.

A new set of 20 Multi-InDel markers for forensic application.

Insertion/deletion markers (InDels) become an important marker for forensic medicine because of their compatible typing techniques with STRs and lower mutation rates. Recent years, a new kind of DNA marker named Multi-InDel was reported as characterized by two or more tightly linked InDel loci within a short length of physical position, usually 200-300 nucleotides. Many pieces of research showed that Multi-InDels had excellent application values in ancestry inference and forensic medicine. Since the identical number of insertion/deletion nucleotides of the InDel markers that composing the Multi-InDel marker, the genotypes of most reported Multi-InDels could not be directly typed by capillary electrophoresis (CE) due to the lack of length discrepancy among the composing InDel sequence. In this study, we applied a typing system of 20 Multi-InDels including 41 InDels, whose genotypes could be deduced by CE and assessed their potential applications in forensic medicine. A total of 200 unrelated Chinese Han individuals and five mother-child-father trios with proven paternity with one STR locus transmission incompatibilities from Shanxi province were genotyped by the multiplex system. The results showed that a total of 70 specific alleles were observed, more than three alleles were observed in 19 loci and seven alleles were observed in one locus. The combined probability of exclusion and the combined power of discrimination were 0.992 and 0.99999999993, respectively. This study demonstrates their potential usefulness for individual identification and paternity tests. The development of Multi-InDels provided another genetic tool inherent in higher polymorphic and lower mutation rates.

Effect of indel variants within the sorting nexin 29 (SNX29) gene on growth traits of goats

The sorting nexin 29 gene (SNX29) is a well-known regulator of myocyte differentiation and proliferation. In this work, two indels (17-bp and 21-bp) were identified in the goat SNX29 gene, and their effects on the growth traits of 1,759 Shaanbei white cashmere (SBWC) goats were analyzed. Both indels had three genotypes [homozygote wild type (II), heterozygote (ID), and homozygote mutation (DD)] and displayed medium genetic diversity (0.25 < polymorphism information content (PIC) < 0.50) in the population. The 17-bp indel was significantly associated with chest width (p = 0.009), body weight (p = 0.021), and chest depth (p = 0.032), with the II genotype dominant. The 21-bp indel was significantly associated with chest width (p = 0.001), chest depth (p = 4.8E-5), heart girth (p = 0.007), and hip width (p = 0.002). Because the two indels were in the upstream (17-bp) and intron (21-bp) regions of the SNX29 gene, transcription factor binding sites were predicted. The IRF5 and MYC could bind with the 17-bp indel and 21-bp indel sequences, respectively. This study indicates that SNX29 is a promising candidate gene that can be used to improve meat production in goat breeding.

RNAIndel: discovering somatic coding indels from tumor RNA-Seq data.

Motivation Reliable identification of expressed somatic insertions/deletions (indels) is an unmet need due to artifacts generated in PCR-based RNA-Seq library preparation and the lack of normal RNA-Seq data, presenting analytical challenges for discovery of somatic indels in tumor transcriptome. Results We present RNAIndel, a tool for predicting somatic, germline and artifact indels from tumor RNA-Seq data. RNAIndel leverages features derived from indel sequence context and biological effect in a machine-learning framework. Except for tumor samples with microsatellite instability, RNAIndel robustly predicts 88-100% of somatic indels in five diverse test datasets of pediatric and adult cancers, even recovering subclonal (VAF range 0.01-0.15) driver indels missed by targeted deep-sequencing, outperforming the current best-practice for RNA-Seq variant calling which had 57% sensitivity but with 14 times more false positives. Availability and implementation RNAIndel is freely available at https://github.com/stjude/RNAIndel. Supplementary information Supplementary data are available at Bioinformatics online.

High throughput genotyping of structural variations in a complex plant genome using an original Affymetrix\xae axiom\xae array

BackgroundInsertions/deletions (InDels) and more specifically presence/absence variations (PAVs) are pervasive in several species and have strong functional and phenotypic effect by removing or drastically modifying genes. Genotyping of such variants on large panels remains poorly addressed, while necessary for approaches such as association mapping or genomic selection.ResultsWe have developed, as a proof of concept, a new high-throughput and affordable approach to genotype InDels. We first identified 141,000 InDels by aligning reads from the B73 line against the genome of three temperate maize inbred lines (F2, PH207, and C103) and reciprocally. Next, we designed an Affymetrix® Axiom® array to target these InDels, with a combination of probes selected at breakpoint sites (13%) or within the InDel sequence, either at polymorphic (25%) or non-polymorphic sites (63%) sites. The final array design is composed of 662,772 probes and targets 105,927 InDels, including PAVs ranging from 35 bp to 129kbp. After Affymetrix® quality control, we successfully genotyped 86,648 polymorphic InDels (82% of all InDels interrogated by the array) on 445 maize DNA samples with 422,369 probes. Genotyping InDels using this approach produced a highly reliable dataset, with low genotyping error (~ 3%), high call rate (~ 98%), and high reproducibility (> 95%). This reliability can be further increased by combining genotyping of several probes calling the same InDels (< 0.1% error rate and > 99.9% of call rate for 5 probes). This “proof of concept” tool was used to estimate the kinship matrix between 362 maize lines with 57,824 polymorphic InDels. This InDels kinship matrix was highly correlated with kinship estimated using SNPs from Illumina 50 K SNP arrays.ConclusionsWe efficiently genotyped thousands of small to large InDels on a sizeable number of individuals using a new Affymetrix® Axiom® array. This powerful approach opens the way to studying the contribution of InDels to trait variation and heterosis in maize. The approach is easily extendable to other species and should contribute to decipher the biological impact of InDels at a larger scale.

New Genetic Markers for Molecular Typing of Bacillus anthracis Strains

Objective : Identification of new markers for the molecular typing of Bacillus anthracis. Materials and methods . The genomes of 16 B. anthracis strains from the collection of the Stavropol Research Anti-Plague Institute, 11 B. anthracis strains and 5 strains of Bacillus cereus from GenBank were investigated. The methods of in vitro and in silico analysis of canonical and whole-genome single nucleotide polymorphisms (SNP), genome regions with variable number of tandem repeats (VNTR) were used for the analysis. Results and discussion . It has been established that there are deletions and (or) SNPs in some of B. anthracis strains of the main genetic lineage B, within the homologous genes of the tri-cistronic operon gerH, which encodes spore germination proteins. gerA genes contain the Bams34 VNTR locus, the sizes of genes in different strains vary due to the different number of tandem repeats and the presence of indels, which suggests the variability of GerA spore germination proteins. In the area of reverse primer annealing, some of them have several SNPs or deletions, which makes impossible PCR amplification of the Bams34 locus. Previously not described VNTR locus, SNPs and indels in sequences of plasmids pXO1 and pXO2, as well as SNP in chromosomal gene of glycerol-3-phosphate transporter were identified. Two pairs of PCR primers for the variable regions of the plasmids were designed. VNTR-locus, SNP and indels in sequences of plasmids pXO1 and pXO2 are suitable genetic markers for the differentiation of typical virulent diplasmid strains belonging to the main genetic lineages of B. anthracis A, B and C. The allele T of SNP within chromosomal glpT gene is specific for one of two strains isolated during the outbreak of anthrax and distinguishes it from all other strains of B. anthracis.

RNAIndel: discovering somatic coding indels from tumor RNA-Seq data.

Reliable identification of expressed somatic insertions/deletions (indels) is an unmet need due to artifacts generated in PCR-based RNA-Seq library preparation and the lack of normal RNA-Seq data, presenting analytical challenges for discovery of somatic indels in tumor transcriptome. We present RNAIndel, a tool for predicting somatic, germline and artifact indels from tumor RNA-Seq data. RNAIndel leverages features derived from indel sequence context and biological effect in a machine-learning framework. Except for tumor samples with microsatellite instability, RNAIndel robustly predicts 88-100% of somatic indels in five diverse test datasets of pediatric and adult cancers, even recovering subclonal (VAF range 0.01-0.15) driver indels missed by targeted deep-sequencing, outperforming the current best-practice for RNA-Seq variant calling which had 57% sensitivity but with 14 times more false positives. RNAIndel is freely available at https://github.com/stjude/RNAIndel. Supplementary data are available at Bioinformatics online.

A multiallelic indel in the promoter region of the Cyclin-dependent kinase inhibitor 3 gene is significantly associated with body weight and carcass traits in chickens.

Many studies have reported that cyclin-dependent kinase inhibitor 3 (CDKN3) is involved in the cell cycle. However, the function of CDKN3 has not been well elucidated in organisms. In this study, a multiallelic indel caused by a 19-bp fragment and a 2 × 19bp fragment was shown for the first time to be inserted into the promoter of the CDKN3 gene in 1994 chickens from 9 different breeds. In addition, 6 genotypes (C5C5, C4C4, C3C3, C4C5, C3C4, and C3C5) were observed (C3C3, C4C4, C5C5 have 3 × 19bp, 4 × 19bp, and 5 × 19bp, respectively). Among these genotypes, the C4C4 genotype was the most dominant genotype in 9 breeds. The results of χ2 analysis of CDKN3 gene in different breeds showed that there were significant differences in the distribution of genotypes among different cultivars (P < 0.01). In addition, association study with F2 chicken resource population which produced by Anka and Gushi chickens showed that the C3C4 genotypes had the greatest semi-evisceration weight (SEW, 1163.94 ± 46.84), evisceration weight (EW, 964.15 ± 41.16), head weight (HW, 45.55 ± 1.43), claw weight (CW, 63.42±2.86), wing weight (WW, 129.15±5.48), liver weight (LW, 29.96±1.27), carcass weight (cW, 1286.96±49.53), weight at 10 (1190.68±45.68) and 12 (1430.65±54.45) wk, followed by C3C3, C4C4, C5C5, C4C5, whereas C3C5 genotypes having the lowest SEW (989.21±47.71), EW (841.38±40.55), HW (41.03±1.46), CW (54.36±2.81), WW (116.31±5.39), LW (27.31±1.25), cW (1093.29±49.99), weight at 10 (1036.10±44.99) and 12 (1246.28±53.59) wk. Expression levels of CDKN3 in breast muscle of chickens with C4C4 (0.72±0.02), C3C3 (0.95±0.41), and C4C5 (0.74±0.13) genotypes were significantly lower than those with C5C5 (1.80±0.01) and C3C5 (2.14±0.17) genotypes (P < 0.05). In conclusion, we investigated the effect of a multiallelic indel in the CDKN3 gene on the economic traits of chickens, and this indel was significantly associated with growth and carcass traits in chickens. Collectively, our findings provide useful information about the repeat sequence indel in the promoter region of the CDKN3 gene as a potential molecular marker for chicken breeding.

Contributions of substitutions and indels to the structural variations in ancient protein superfamilies

BackgroundQuantitative evaluation of protein structural evolution is important for our understanding of protein biological functions and their evolutionary adaptation, and is useful in guiding protein engineering. However, compared to the models for sequence evolution, the quantitative models for protein structural evolution received less attention. Ancient protein superfamilies are often considered versatile, allowing genetic and functional diversifications during long-term evolution. In this study, we investigated the quantitative impacts of sequence variations on the structural evolution of homologues in 68 ancient protein superfamilies that exist widely in sequenced eukaryotic, bacterial and archaeal genomes.ResultsWe found that the accumulated structural variations within ancient superfamilies could be explained largely by a bilinear model that simultaneously considers amino acid substitution and insertion/deletion (indel). Both substitutions and indels are essential for explaining the structural variations within ancient superfamilies. For those ancient superfamilies with high bilinear multiple correlation coefficients, the influence of each unit of substitution or indel on structural variations is almost constant within each superfamily, but varies greatly among different superfamilies. The influence of each unit indel on structural variations is always larger than that of each unit substitution within each superfamily, but the accumulated contributions of indels to structural variations are lower than those of substitutions in most superfamilies. The total contributions of sequence indels and substitutions (46% and 54%, respectively) to the structural variations that result from sequence variations are slightly different in ancient superfamilies.ConclusionsStructural variations within ancient protein superfamilies accumulated under the significantly bilinear influence of amino acid substitutions and indels in sequences. Both substitutions and indels are essential for explaining the structural variations within ancient superfamilies. For those structural variations resulting from sequence variations, the total contribution of indels is slightly lower than that of amino acid substitutions. The regular clock exists not only in protein sequences, but also probably in protein structures.

Genetic Variation in the H19-IGF2 Cluster Might Confer Risk of Developing Impaired Renal Function.

The H19-IGF2 imprinted gene region could be implicated in the risk of developing impaired renal function (IRF). Our aim was to determine the association of several common H19-IGF2 variants and IRF in a cohort of elderly healthy individuals. The study involved 675 individuals >65 years of age, 184 with type 2 diabetes mellitus (T2DM), and 105 with IRF (estimated glomerular filtration rate [eGFR] <60). They were genotyped for two common H19 single nucleotide polymorphisms (SNPs) (rs2839698 and rs10732516), one H19-IGF2 intergenic indel (rs201858505), and one indel in the 3'UTR of the IGF2. For the H19 SNPs, we also determined the allele present in the methylated chromosome through genotyping the DNA digested with a methylation-sensitive endonuclease. None of the four H19-IGF2 variants was associated with IRF in our cohort. We found a significantly higher frequency of the 3'UTR IGF2 deletion (D) in the eGFR <60 group (p = 0.01; odds ratio = 1.16, 95% confidence interval = 1.10-2.51). This association was independent of age and T2DM, two strong predictors of IRF. In conclusion, a common indel variant in the 3'UTR of the IGF2 gene was associated with the risk of IRF. This association could be explained by the role of IGF2 in podocyte survival, through regulation of IGF2 expression by differential binding of miRNAs to the indel sequences. Functional studies should be necessary to clarify this issue.

RIFRAF: a frame-resolving consensus algorithm.

Protein coding genes can be studied using long-read next generation sequencing. However, high rates of indel sequencing errors are problematic, corrupting the reading frame. Even the consensus of multiple independent sequence reads retains indel errors. To solve this problem, we introduce Reference-Informed Frame-Resolving multiple-Alignment Free template inference algorithm (RIFRAF), a sequence consensus algorithm that takes a set of error-prone reads and a reference sequence and infers an accurate in-frame consensus. RIFRAF uses a novel structure, analogous to a two-layer hidden Markov model: the consensus is optimized to maximize alignment scores with both the set of noisy reads and with a reference. The template-to-reads component of the model encodes the preponderance of indels, and is sensitive to the per-base quality scores, giving greater weight to more accurate bases. The reference-to-template component of the model penalizes frame-destroying indels. A local search algorithm proceeds in stages to find the best consensus sequence for both objectives. Using Pacific Biosciences SMRT sequences from an HIV-1 env clone, NL4-3, we compare our approach to other consensus and frame correction methods. RIFRAF consistently finds a consensus sequence that is more accurate and in-frame, especially with small numbers of reads. It was able to perfectly reconstruct over 80% of consensus sequences from as few as three reads, whereas the best alternative required twice as many. RIFRAF is able to achieve these results and keep the consensus in-frame even with a distantly related reference sequence. Moreover, unlike other frame correction methods, RIFRAF can detect and keep true indels while removing erroneous ones. RIFRAF is implemented in Julia, and source code is publicly available at https://github.com/MurrellGroup/Rifraf.jl. Supplementary data are available at Bioinformatics online.

The Complete Chloroplast Genome Sequences of Aconitum pseudolaeve and Aconitum longecassidatum, and Development of Molecular Markers for Distinguishing Species in the Aconitum Subgenus Lycoctonum.

Aconitum pseudolaeve Nakai and Aconitum longecassidatum Nakai, which belong to the Aconitum subgenus Lycoctonum, are distributed in East Asia and Korea. Aconitum species are used in herbal medicine and contain highly toxic components, including aconitine. A. pseudolaeve, an endemic species of Korea, is a commercially valuable material that has been used in the manufacture of cosmetics and perfumes. Although Aconitum species are important plant resources, they have not been extensively studied, and genomic information is limited. Within the subgenus Lycoctonum, which includes A. pseudolaeve and A. longecassidatum, a complete chloroplast (CP) genome is available for only one species, Aconitum barbatum Patrin ex Pers. Therefore, we sequenced the complete CP genomes of two Aconitum species, A. pseudolaeve and A. longecassidatum, which are 155,628 and 155,524 bp in length, respectively. Both genomes have a quadripartite structure consisting of a pair of inverted repeated regions (51,854 and 52,108 bp, respectively) separated by large single-copy (86,683 and 86,466 bp) and small single-copy (17,091 and 16,950 bp) regions similar to those in other Aconitum CP genomes. Both CP genomes consist of 112 unique genes, 78 protein-coding genes, 4 ribosomal RNA (rRNA) genes, and 30 transfer RNA (tRNA) genes. We identified 268 and 277 simple sequence repeats (SSRs) in A. pseudolaeve and A. longecassidatum, respectively. We also identified potential 36 species-specific SSRs, 53 indels, and 62 single-nucleotide polymorphisms (SNPs) between the two CP genomes. Furthermore, a comparison of the three Aconitum CP genomes from the subgenus Lycoctonum revealed highly divergent regions, including trnK-trnQ, ycf1-ndhF, and ycf4-cemA. Based on this finding, we developed indel markers using indel sequences in trnK-trnQ and ycf1-ndhF. A. pseudolaeve, A. longecassidatum, and A. barbatum could be clearly distinguished using the novel indel markers AcoTT (Aconitum trnK-trnQ) and AcoYN (Aconitum ycf1-ndhF). These two new complete CP genomes provide useful genomic information for species identification and evolutionary studies of the Aconitum subgenus Lycoctonum.

Differential evolution-simulated annealing for multiple sequence alignment

Multiple sequence alignments (MSA) are used in the analysis of molecular evolution and sequence structure relationships. In this paper, a hybrid algorithm, Differential Evolution - Simulated Annealing (DESA) is applied in optimizing multiple sequence alignments (MSAs) based on structural information, non-gaps percentage and totally conserved columns. DESA is a robust algorithm characterized by self-organization, mutation, crossover, and SA-like selection scheme of the strategy parameters. Here, the MSA problem is treated as a multi-objective optimization problem of the hybrid evolutionary algorithm, DESA. Thus, we name the algorithm as DESA-MSA. Simulated sequences and alignments were generated to evaluate the accuracy and efficiency of DESA-MSA using different indel sizes, sequence lengths, deletion rates and insertion rates. The proposed hybrid algorithm obtained acceptable solutions particularly for the MSA problem evaluated based on the three objectives.