Copy number variants impact phenotype-genotype relationships for adaptation of industrial yeast Saccharomyces cerevisiae.

The industrial yeast Saccharomyces cerevisiae possesses a plastic genome enabling its adaptation to varied environment conditions. A more robust ethanologenic industrial yeast strain NRRL Y-50049 was obtained through laboratory adaptation that is resistant to 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF), a major class of toxic chemicals associated with lignocellulose-to-biofuel conversion. A significant amount of knowledge has been achieved in characterizing its tolerant phenotypes and molecular mechanisms of the resistance. Recent findings on a limited number of nonsynonymous SNP (single nucleotide polymorphism) detected in NRRL Y-50049 compared with its progenitor NRRL Y-12632 raised doubt of SNP roles in the tolerance adaptation. The genotype-phenotype relationship for yeast adaptation to the toxic chemicals is yet unclear. Here, we examine copy number variant (CNV) of the adapted strain NRRL Y-50049 to address phenotype-genotype relationships. As a background information, CNV of model strain S288C of the reference genome was also examined versus the industrial-type strain NRRL Y-12632. More than 200 CNVs, mostly duplication events, were detected in NRRL Y-12632 compared with the laboratory model strain S288C. Such enriched genetic background supports its more diversified phenotype response for the industrial yeast than the laboratory strain S288C. Comparing the two industrial strains, we found extra nine CNVs in the mitochondrial genome and 28 CNVs in the nuclear genome of NRRL Y-50049 versus its progenitor NRRL Y-12632. Continued DNA recombination event and high rate of CNV observed in NRRL Y-50049 versus its progenitor suggests that CNV is more impactful than SNP in association with phenotype-genotype relationships of yeast adaptation to the toxic chemical stress. COX1 and COB loci were defined as DNA recombination hotspots in the mitochondrial genome for the industrial yeast based on the high frequency of CNVs observed in these loci. KEY POINTS: • COX1 and COB loci are identified as DNA recombination hotspots for the industrial yeast. • The industrial yeast type strain NRRL Y-12632 possesses more CNVs vs the reference genome S288C. • CNV is more important than SNP on phenotype-genotype relationships for yeast adaptation.

A glimpse of potential transposable element impact on adaptation of the industrial yeast Saccharomyces cerevisiae.

The adapted industrial yeast strain Saccharomyces cerevisiae NRRL Y-50049 is able to in situ detoxify major toxic aldehyde compounds derived from sugar conversion of lignocellulosic biomass while producing ethanol. Pathway-based studies on its mechanisms of tolerance have been reported previously, however, little is known about transposable element (TE) involvement in its adaptation to inhibitory compounds. This work presents a comparative dynamic transcription expression analysis in response to a toxic treatment between Y-50049 and its progenitor, an industrial type strain NRRL Y-12632, using a time-course study. At least 77 TEs from Y-50049 showed significantly increased expression compared with its progenitor, especially during the late lag phase. Sequence analysis revealed significant differences in TE sequences between the two strains. Y-50049 was also found to have a transposons of yeast 2 (Ty2) long terminal repeat-linked YAT1 gene showing significantly higher copy number changes than its progenitor. These results raise awareness of potential TE involvement in the adaptation of industrial yeast to the tolerance of toxic chemicals.

Protein expression analysis revealed a fine-tuned mechanism of in situ detoxification pathway for the tolerant industrial yeast Saccharomyces cerevisiae.

Inhibitory compounds liberated from lignocellulose pretreatment are representative toxic chemicals that repress microbial growth and metabolism. A tolerant strain of the industrial yeast Saccharomyces cerevisiae is able to detoxify a major class of toxic compounds while producing ethanol. Knowledge on the yeast tolerance was mostly obtained by gene expression analysis and limited protein expression evidence is yet available underlying the yeast adaptation. Here we report a comparative protein expression profiling study on Y-50049, a tolerant strain compared with its parental industrial type strain Y-12632. We found a distinctive protein expression of glucose-6-phosphate dehydrogenase (Zwf1) in Y-50049 but not in Y-12632, in the relatively conserved glycolysis and pentose phosphate pathway (PPP) in response to a combinational challenge of 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF). A group of proteins with aldehyde reduction activity was uniquely induced expressed in Y-50049 but not in Y-12632. Such evidence allowed fine-tuning a mechanism of the renovated in situ detoxification by Y-50049. As the key protein, Zwf1 drove the glucose metabolism in favor of the oxidative branch of the PPP facilitating in situ detoxification of the toxic chemicals by Y-50049. The activated expression of Zwf1 generated the essential cofactor nicotinamide adenine dinucleotide phosphate (NADPH) enabling reduction of furfural and HMF through a group of aldehyde reduction enzymes. In return, the activate aldehyde reductions released desirable feedbacks of NADP+ stimulating continued oxidative activity of Zwf1. Thus, a well-maintained cofactor regeneration cycle was established to restore the cofactor imbalance caused by furfural-HMF. Challenges and perspectives on adaptation of significantly differential expressions of ribosomal proteins and other unique proteins are also discussed.

NucAmino: a nucleotide to amino acid alignment optimized for virus gene sequences.

BACKGROUND: Current nucleotide-to-amino acid alignment software programs were developed primarily for detecting gene exons within eukaryotic genomes and were therefore optimized for speed across long genetic sequences. We developed a nucleotide-to-amino acid alignment program NucAmino optimized for virus sequencing. RESULTS: NucAmino is an open source program written in the high-level language Go. NucAmino is more likely to align codons flush with a reference sequence's amino acids and can be modified to facilitate the placement of insertions and deletions at specific positions. We compared NucAmino to the nucleotide to amino acid alignment program Local Alignment Program (LAP) using 115,118 human immunodeficiency virus type 1 (HIV-1) protease, reverse transcriptase, and integrase sequences-three genes that are commonly sequenced in clinical laboratories. Discordances between NucAmino and LAP occurred in 512 (16.9%) of the 3,029 sequences containing gaps but in none of 112,910 sequences without gaps. For 242 of the sequences with discordances, NucAmino produced an alignment that was preferable to that found by LAP in that it was more likely to codon align insertions and deletions and to facilitate the placement of an important drug-resistance associated insertion at the position at which most laboratories expect it to occur. CONCLUSIONS: NucAmino is a nucleotide-to-amino acid alignment program with several advantages for clinical laboratories performing virus sequencing compared with older programs designed for gene finding.

Genetic architecture and evolution of the mating type locus in fusaria that cause soybean sudden death syndrome and bean root rot.

Fusarium tucumaniae is the only known sexually reproducing species among the seven closely related fusaria that cause soybean sudden death syndrome (SDS) or bean root rot (BRR). In a previous study, laboratory mating of F. tucumaniae yielded recombinant ascospore progeny but required two mating-compatible strains, indicating that it is heterothallic. To assess the reproductive mode of the other SDS and BRR fusaria, and their potential for mating, whole-genome sequences of two SDS and one BRR pathogen were analyzed to characterize their mating type (MAT) loci. This bioinformatic approach identified a MAT1-1 idiomorph in F. virguliforme NRRL 22292 and MAT1-2 idiomorphs in F. tucumaniae NRRL 34546 and F. azukicola NRRL 54364. Alignments of the MAT loci were used to design PCR primers within the conserved regions of the flanking genes APN1 and SLA2, which enabled primer walking to obtain nearly complete sequences of the MAT region for six MAT1-1 and five MAT1-2 SDS/BRR fusaria. As expected, sequences of the highly divergent 4.7 kb MAT1-1 and 3.7 kb MAT1-2 idiomorphs were unalignable. However, sequences of the respective idiomorphs and those that flank MAT1-1 and MAT1-2 were highly conserved. In addition to three genes at MAT1-1 (MAT1-1-1, MAT1-1-2, MAT1-1-3) and two at MAT1-2 (MAT1-2-1, MAT1-2-3), the MAT loci of the SDS/BRR fusaria also include a putative gene predicted to encode for a 252 amino acid protein of unknown function. Alignments of the MAT1-1-3 and MAT1-2-1 sequences were used to design a multiplex PCR assay for the MAT loci. This assay was used to screen DNA from 439 SDS/BRR isolates, which revealed that each isolate possessed MAT1-1 or MAT1-2, consistent with heterothallism. Both idiomorphs were represented among isolates of F. azukicola, F. brasiliense, F. phaseoli and F. tucumaniae, whereas isolates of F. virguliforme and F. cuneirostrum were only MAT1-1 and F. crassistipitatum were only MAT1-2. Finally, nucleotide sequence data from the RPB1 and RPB2 genes were used to date the origin of the SDS/BRR group, which was estimated to have occurred about 0.75 Mya (95% HPD interval: 0.27, 1.68) in the mid-Pleistocene, long before the domestication of the common bean or soybean.

Differences in epidemiological and clinical features between adult and pediatric tracheobronchial tuberculosis patients in Southwest China.

Background: Tracheobronchial tuberculosis (TBTB) is a common form of extrapulmonary tuberculosis that affects the tracheobronchial tree. However, the mechanism has not been fully elucidated. Comparisons of clinical characteristics in various age groups can aid in the understanding of TBTB. Methods: This retrospective study was conducted at the Public Health Clinical Center of Chengdu between July 2017 and December 2021, including adults and children with TBTB. Clinical data were extracted from medical records. T/T' test, Mann-Whitney U test, Chi-square test, or Fisher's exact test were used in this study. Results: This study enrolled 347 patients with TBTB (175 adults and 172 children). Adult females were more susceptible to TBTB, whereas gender-based differences were not observed in children. Children had a higher occurrence of irritant dry cough and fever, and acute hematogenous disseminated PTB, and specific types of EPTB, but a shorter interval before diagnosis, and lower diagnostic yields compared to adults (P < 0.05). Adults presented more extensive lung lesions and cavitations as compared to children. Granulation hyperplasia and lymph fistula were more frequently observed in children, as well as airway stenosis, but less severe. Conclusions: The study revealed important variations exist in multiple respects between adults and children with TBTB.

Sequence-specific sequence comparison using pairwise statistical significance.

There has been a deluge of biological sequence data in the public domain, which makes sequence comparison one of the most fundamental computational problems in bioinformatics. The biologists routinely use pairwise alignment programs to identify similar, or more specifically, related sequences (having common ancestor). It is a well-known fact that almost everything in bioinformatics depends on the inter-relationship between sequence, structure, and function (all encapsulated in the term relatedness), which is far from being well understood. The potential relatedness of two sequences is better judged by statistical significance of the alignment score rather than by the alignment score alone. This chapter presents a summary of recent advances in accurately estimating statistical significance of pairwise local alignment for the purpose of identifying related sequences, by making the sequence comparison process more sequence specific. Comparison of using pairwise statistical significance to rank database sequences, with well-known database search programs like BLAST, PSI-BLAST, and SSEARCH, is also presented. As expected, the sequence-comparison performance (evaluated in terms of retrieval accuracy) improves significantly as the sequence comparison process is made more and more sequence specific. Shortcomings of currently used approaches and some potentially useful directions for future work are also presented.

Tightly linked Rps12 and Rps13 genes provide broad-spectrum Phytophthora resistance in soybean.

The Phytophtora root and stem rot is a serious disease in soybean. It is caused by the oomycete pathogen Phytophthora sojae. Growing Phytophthora resistant cultivars is the major method of controlling this disease. Resistance is race- or gene-specific; a single gene confers immunity against only a subset of the P. sojae isolates. Unfortunately, rapid evolution of new Phytophthora sojae virulent pathotypes limits the effectiveness of an Rps ("resistance to Phytophthora sojae") gene to 8-15 years. The current study was designed to investigate the effectiveness of Rps12 against a set of P. sojae isolates using recombinant inbred lines (RILs) that contain recombination break points in the Rps12 region. Our study revealed a unique Rps gene linked to the Rps12 locus. We named this novel gene as Rps13 that confers resistance against P. sojae isolate V13, which is virulent to recombinants that contains Rps12 but lack Rps13. The genetic distance between the two Rps genes is 4 cM. Our study revealed that two tightly linked functional Rps genes with distinct race-specificity provide broad-spectrum resistance in soybean. We report here the molecular markers for incorporating the broad-spectrum Phytophthora resistance conferred by the two Rps genes in commercial soybean cultivars.

PSIBLAST_PairwiseStatSig: reordering PSI-BLAST hits using pairwise statistical significance.

We present an add-on to BLAST and PSI-BLAST programs to reorder their hits using pairwise statistical significance. Using position-specific substitution matrices to estimate pairwise statistical significance has been recently shown to give promising results in terms of retrieval accuracy, which motivates its use to refine PSI-BLAST results, since PSI-BLAST also constructs a position-specific substitution matrix for the query sequence during the search. The obvious advantage of the approach is more accurate estimates of statistical significance because of pairwise statistical significance, along with the advantage of BLAST/PSI-BLAST in terms of speed.

Pairwise statistical significance of local sequence alignment using multiple parameter sets and empirical justification of parameter set change penalty.

BACKGROUND: Accurate estimation of statistical significance of a pairwise alignment is an important problem in sequence comparison. Recently, a comparative study of pairwise statistical significance with database statistical significance was conducted. In this paper, we extend the earlier work on pairwise statistical significance by incorporating with it the use of multiple parameter sets. RESULTS: Results for a knowledge discovery application of homology detection reveal that using multiple parameter sets for pairwise statistical significance estimates gives better coverage than using a single parameter set, at least at some error levels. Further, the results of pairwise statistical significance using multiple parameter sets are shown to be significantly better than database statistical significance estimates reported by BLAST and PSI-BLAST, and comparable and at times significantly better than SSEARCH. Using non-zero parameter set change penalty values give better performance than zero penalty. CONCLUSION: The fact that the homology detection performance does not degrade when using multiple parameter sets is a strong evidence for the validity of the assumption that the alignment score distribution follows an extreme value distribution even when using multiple parameter sets. Parameter set change penalty is a useful parameter for alignment using multiple parameter sets. Pairwise statistical significance using multiple parameter sets can be effectively used to determine the relatedness of a (or a few) pair(s) of sequences without performing a time-consuming database search.

Dynamic use of multiple parameter sets in sequence alignment.

The level of conservation between two homologous sequences often varies among sequence regions; functionally important domains are more conserved than the remaining regions. Thus, multiple parameter sets should be used in alignment of homologous sequences with a stringent parameter set for highly conserved regions and a moderate parameter set for weakly conserved regions. We describe an alignment algorithm to allow dynamic use of multiple parameter sets with different levels of stringency in computation of an optimal alignment of two sequences. The algorithm dynamically considers various candidate alignments, partitions each candidate alignment into sections, and determines the most appropriate set of parameter values for each section of the alignment. The algorithm and its local alignment version are implemented in a computer program named GAP4. The local alignment algorithm in GAP4, that in its predecessor GAP3, and an ordinary local alignment program SIM were evaluated on 257,716 pairs of homologous sequences from 100 protein families. On 168,475 of the 257,716 pairs (a rate of 65.4%), alignments from GAP4 were more statistically significant than alignments from GAP3 and SIM.

HPC: Hierarchical phylogeny construction.

Rapid improvements in DNA sequencing technology have resulted in long genome sequences for a large number of similar isolates with a wide range of single nucleotide polymorphism (SNP) rates, where some isolates can have thousands of times lower SNP rates than others. Genome sequences of this kind are a challenge to existing methods for construction of phylogenetic trees. We address the issues by developing a hierarchical approach to phylogeny construction. In this method, the construction is performed at multiple levels, where at each level, groups of isolates with similar levels of similarity are identified and their phylogenetic trees are constructed. Time savings are achieved by using a sufficiently large number of columns from the input alignment, instead of all its columns. Our results show that the new approach is 20-60 times more efficient than existing programs and more accurate in situations where highly similar isolates have a wide range of SNP rates.

Genome wide association study identifies novel single nucleotide polymorphic loci and candidate genes involved in soybean sudden death syndrome resistance.

Fusarium virguliforme is a soil borne root pathogen that causes sudden death syndrome (SDS) in soybean [Glycine max (L.) Merrill]. Once the fungus invades the root xylem tissues, the pathogen secretes toxins that cause chlorosis and necrosis in foliar tissues leading to defoliation, flower and pod drop and eventually death of plants. Resistance to F. virguliforme in soybean is partial and governed by over 80 quantitative trait loci (QTL). We have conducted genome-wide association study (GWAS) for a group of 254 plant introductions lines using a panel of approximately 30,000 SNPs and identified 19 single nucleotide polymorphic loci (SNPL) that are associated with 14 genomic regions encoding foliar SDS and eight SNPL associated with seven genomic regions for root rot resistance. Of the identified 27 SNPL, six SNPL for foliar SDS resistance and two SNPL for root rot resistance co-mapped to previously identified QTL for SDS resistance. This study identified 13 SNPL associated with eight novel genomic regions containing foliar SDS resistance genes and six SNPL with five novel regions for root-rot resistance. This study identified five genes carrying nonsynonymous mutations: (i) three of which mapped to previously identified QTL for foliar SDS resistance and (ii) two mapped to two novel regions containing root rot resistance genes. Of the three genes mapped to QTL for foliar SDS resistance genes, two encode LRR-receptors and third one encodes a novel protein with unknown function. Of the two genes governing root rot resistance, Glyma.01g222900.1 encodes a soybean-specific LEA protein and Glyma.10g058700.1 encodes a heparan-alpha-glucosaminide N-acetyltransferase. In the LEA protein, a conserved serine residue was substituted with asparagine; and in the heparan-alpha-glucosaminide N-acetyltransferase, a conserved histidine residue was substituted with an arginine residue. Such changes are expected to alter functions of these two proteins regulated through phosphorylation. The five genes with nonsynonymous mutations could be considered candidate SDS resistance genes and should be suitable molecular markers for breeding SDS resistance in soybean. The study also reports desirable plant introduction lines and novel genomic regions for enhancing SDS resistance in soybean.

Sequence alignment with an appropriate substitution matrix.

A widely used algorithm for computing an optimal local alignment between two sequences requires a parameter set with a substitution matrix and gap penalties. It is recognized that a proper parameter set should be selected to suit the level of conservation between sequences. We describe an algorithm for selecting an appropriate substitution matrix at given gap penalties for computing an optimal local alignment between two sequences. In the algorithm, a substitution matrix that leads to the maximum alignment similarity score is selected among substitution matrices at various evolutionary distances. The evolutionary distance of the selected substitution matrix is defined as the distance of the computed alignment. To show the effects of gap penalties on alignments and their distances and help select appropriate gap penalties, alignments and their distances are computed at various gap penalties. The algorithm has been implemented as a computer program named SimDist. The SimDist program was compared with an existing local alignment program named SIM for finding reciprocally best-matching pairs (RBPs) of sequences in each of 100 protein families, where RBPs are commonly used as an operational definition of orthologous sequences. SimDist produced more accurate results than SIM on 50 of the 100 families, whereas both programs produced the same results on the other 50 families. SimDist was also used to compare three types of substitution matrices in scoring 444,461 pairs of homologous sequences from the 100 families.

Application of a superword array in genome assembly.

We introduce a data structure called a superword array for finding quickly matches between DNA sequences. The superword array possesses some desirable features of the lookup table and suffix array. We describe simple algorithms for constructing and using a superword array to find pairs of sequences that share a unique superword. The algorithms are implemented in a genome assembly program called PCAP.REP for computation of overlaps between reads. Experimental results produced by PCAP.REP and PCAP on a whole-genome dataset show that PCAP.REP produced a more accurate and contiguous assembly than PCAP.

Genomic diversity of Taylorella equigenitalis introduced into the United States from 1978 to 2012.

Contagious equine metritis is a disease of worldwide concern in equids. The United States is considered to be free of the disease although sporadic outbreaks have occurred over the last few decades that were thought to be associated with the importation of horses. The objective of this study was to create finished, reference quality genomes that characterize the diversity of Taylorella equigenitalis isolates introduced into the USA, and identify their differences. Five isolates of T. equigenitalis associated with introductions into the USA from unique sources were sequenced using both short and long read chemistries allowing for complete assembly and annotation. These sequences were compared to previously published genomes as well as the short read sequences of the 200 isolates in the National Veterinary Services Laboratories' diagnostic repository to identify unique regions and genes, potential virulence factors, and characterize diversity. The 5 genomes varied in size by up to 100,000 base pairs, but averaged 1.68 megabases. The majority of that diversity in size can be explained by repeat regions and 4 main regions of difference, which ranged in size from 15,000 to 45,000 base pairs. The first region of difference contained mostly hypothetical proteins, the second contained the CRISPR, the third contained primarily hemagglutinin proteins, and the fourth contained primarily segments of a type IV secretion system. As expected and previously reported, little evidence of recombination was found within these genomes. Several additional areas of interest were also observed including a mechanism for streptomycin resistance and other virulence factors. A SNP distance comparison of the T. equigenitalis isolates and Mycobacterium tuberculosis complex (MTBC) showed that relatively, T. equigenitalis was a more diverse species than the entirety of MTBC.

MAP2: multiple alignment of syntenic genomic sequences.

We describe a multiple alignment program named MAP2 based on a generalized pairwise global alignment algorithm for handling long, different intergenic and intragenic regions in genomic sequences. The MAP2 program produces an ordered list of local multiple alignments of similar regions among sequences, where different regions between local alignments are indicated by reporting only similar regions. We propose two similarity measures for the evaluation of the performance of MAP2 and existing multiple alignment programs. Experimental results produced by MAP2 on four real sets of orthologous genomic sequences show that MAP2 rarely missed a block of transitively similar regions and that MAP2 never produced a block of regions that are not transitively similar. Experimental results by MAP2 on six simulated data sets show that MAP2 found the boundaries between similar and different regions precisely. This feature is useful for finding conserved functional elements in genomic sequences. The MAP2 program is freely available in source code form at http://bioinformatics.iastate.edu/aat/sas.html for academic use.

Sequence Assembly.

We describe an efficient method for assembling short reads into long sequences. In this method, a hashing technique is used to compute overlaps between short reads, allowing base mismatches in the overlaps. Then an overlap graph is constructed, with each vertex representing a read and each edge representing an overlap. The overlap graph is explored by graph algorithms to find unique paths of reads representing contigs. The consensus sequence of each contig is constructed by computing alignments of multiple reads without gaps. This strategy has been implemented as a short read assembly program called PCAP.Solexa. We also describe how to use PCAP. Solexa in assembly of short reads.

The Effect of the Treatment with Heated Humidified High-Flow Nasal Cannula on Neonatal Respiratory Distress Syndrome in China: A Single-Center Experience.

Background. Noninvasive respiratory support is considered the optimal method of providing assistance to preterm babies with breathing problems, including nasal continuous positive airway pressure (NCPAP) and humidified high flow nasal cannula (HHHFNC). The evidence of the efficacy and safety of HHHFNC used as the primary respiratory support for respiratory distress syndrome (RDS) is insufficient in low- and middle-income countries. Objective. To investigate the effect of heated humidified high flow nasal cannula on neonatal respiratory distress syndrome compared with nasal continuous positive airway pressure. Methods. An observational cross-sectional study was performed at a tertiary neonatal intensive care unit in suburban Wenzhou, China, in the period between January 2014 and December 2015. Results. A total of 128 infants were enrolled in the study: 65 in the HHHFNC group and 63 in the NCPAP group. The respiratory support with HHHFNC was similar to that with NCPAP with regard to the primary outcome. There is no significant difference between two groups in secondary outcomes. Comparing with NCPAP group, the incidence of nasal damage was lower in HHHFNC group. Conclusions. HHHFNC is an effective and well-tolerated strategy as the primary treatment of mild to moderate RDS in preterm infants older than 28 weeks of GA.

Over 20% of human transcripts might form sense-antisense pairs.

The major challenge to identifying natural sense- antisense (SA) transcripts from public databases is how to determine the correct orientation for an expressed sequence, especially an expressed sequence tag sequence. In this study, we established a set of very stringent criteria to identify the correct orientation of each human transcript. We used these orientation-reliable transcripts to create 26 741 transcription clusters in the human genome. Our analysis shows that 22% (5880) of the human transcription clusters form SA pairs, higher than any previous estimates. Our orientation-specific RT-PCR results along with the comparison of experimental data from previous studies confirm that our SA data set is reliable. This study not only demonstrates that our criteria for the prediction of SA transcripts are efficient, but also provides additional convincing data to support the view that antisense transcription is quite pervasive in the human genome. In-depth analyses show that SA transcripts have some significant differences compared with other types of transcripts, with regard to chromosomal distribution and Gene Ontology-annotated categories of physiological roles, functions and spatial localizations of gene products.