Extensive gene duplication in Arabidopsis revealed by pseudo-heterozygosity

Background: It is becoming apparent that genomes harbor massive amounts of structural variation, and that this variation has largely gone undetected for technical reasons. In addition to being inherently interesting, structural variation can cause artifacts when short-read sequencing data are mapped to a reference genome. In particular, spurious SNPs (that do not show Mendelian segregation) may result from mapping of reads to duplicated regions. Recalling SNP using the raw reads of the 1001 Arabidopsis Genomes Project we identified 3.3 million heterozygous SNPs (44% of total). Given that Arabidopsis thaliana (A. thaliana) is highly selfing, we hypothesized that these SNPs reflected cryptic copy number variation, and investigated them further. Results: While genuine heterozygosity should occur in tracts within individuals, heterozygosity at a particular locus is instead shared across individuals in a manner that strongly suggests it reflects segregating duplications rather than actual heterozygosity. Focusing on pseudo-heterozygosity in annotated genes, we used GWAS to map the position of the duplicates, identifying 2500 putatively duplicated genes. The results were validated using de novo genome assemblies from six lines. Specific examples included an annotated gene and nearby transposon that, in fact, transpose together. Conclusions: Our study confirms that most heterozygous SNPs calls in A. thaliana are artifacts, and suggest that great caution is needed when analysing SNP data from short-read sequencing. The finding that 10% of annotated genes are copy-number variables, and the realization that neither gene- nor transposon-annotation necessarily tells us what is actually mobile in the genome suggest that future analyses based on independently assembled genomes will be very informative.

Both Simulation and Sequencing Data Reveal Multiple SARS-CoV-2 Variants Coinfection in COVID-19 Pandemic

SARS-CoV-2 is a single-stranded RNA betacoronavirus with a high mutation rate. The rapidly emerged SARS-CoV-2 variants could increase the transmissibility, aggravate the severity, and even fade the vaccine protection. Although the coinfections of SARS-CoV-2 with other respiratory pathogens have been reported, whether multiple SARS-CoV-2 variants coinfection exists remains controversial. This study collected 12,986 and 4,113 SARS-CoV-2 genomes from the GISAID database on May 11, 2020 (GISAID20May11) and April 1, 2021 (GISAID21Apr1), respectively. With the single-nucleotide variants (SNV) and network clique analysis, we constructed the single-nucleotide polymorphism (SNP) coexistence networks and noted the SNP number of the maximal clique as the coinfection index. The coinfection indices of GISAID20May11 and GISAID21Apr1 datasets were 16 and 34, respectively. Simulating the transmission routes and the mutation accumulations, we discovered the linear relationship between the coinfection index and the coinfected variant number. Based on the linear relationship, we deduced that the COVID-19 cases in the GISAID20May11 and GISAID21Apr1 datasets were coinfected with 2.20 and 3.42 SARS-CoV-2 variants on average. Additionally, we performed Nanopore sequencing on 42 COVID-19 patients to explore the virus mutational characteristics. We found the heterozygous SNPs in 41 COVID-19 cases, which support the coinfection of SARS-CoV-2 variants and challenge the accuracy of phylogenetic analysis. In conclusion, our findings reported the coinfection of SARS-CoV-2 variants in COVID-19 patients, demonstrated the increased coinfected variants number in the epidemic, and provided clues for the prolonged viral shedding and severe symptoms in some cases.

Towards deciphering the structure of long homozygous stretches in cattle genome

p-to-day there is no as universally accepted software tool and threshold parameters to identify runs of homozygosity ( ROH ). The relative position of POH segments in the cattle genome has not been studied extensively. Specific objective of this study was to evaluate the effect of allowed missing and heterozygous SNPs in ROH on their number, on the estimate of inbreeding level, and on structure of ROH segments in the cattle genome. In this study 371 Holsteinized cows from six herds were genotyped with BovineSNP50 array. To identify ROH, the consecutive and sliding runs were carried out with detectRUNS and Plink tools. Neither effect was shown for missing SNPs genotype calls. Allowing even one heterozygous SNP resulted in significant bias of ROH data. Furthermore, the sliding runs identified less ROH than consecutive runs. The mean coefficient of inbreeding across herds was 0.111 ± 0.003 and 0.104 ± 0.003 based on consecutive and sliding runs respectively. It was shown how, using the heterozygous SNPs in ROH, may be possible to derive a distribution of ROH segments in the cow genome. We suggested it was similar to normal distribution. Furthermore, frequency of ROH in the chromosomes did not depend on their length. Of 29 chromosomes, the most abundant with ROH were BTA 14, BTA 7, and BTA 18. The result of this study confirmed more accurately identification of ROH with consecutive runs, uneven their distribution in the cattle genome, significant bias of the data due to allowing heterozygous SNPs in ROH.

ASSESSMENT OF RECENT INBREEDING OF THE HOLSTEINIZED CATTLE

Изучено влияние неучтенных гетерозиготных и негенотипированных SNPs в протяженных гомозиготных сегментах (ROH) на среднее количество и средний суммарный размер ROH-сегментов, а также на вычисление коэффициента инбридинга у голштинизированных черно-пестрых коров. Из 6 племенных заводов Ленинградской области были отобраны 374 коровы. Полногеномное генотипирование их осуществляли чипом BovineSNP50 v2. При выполнении контроля качества однонуклеотидного полиморфизма (SNP) генотипов коров и полногеномном сканировании ROH использовали программу Plink 1.9. После проведения контроля качества SNP-генотипов из 54609 осталось 43298 SNPs. Влияние неучтенных гетерозиготных и негенотипированных SNPs на результаты ROH сканирования оценивали при изменении этих показателей от 0 до 16. Оказалось, что негенотипированные SNPs не влияли на показатели ROH, тогда как количество неучтенных гетерозиготных SNPs существенно влияло на результаты ROH сканирования. Так, по мере увеличения количества неучтенных гетерозиготных SNPs в ROH-сегментах среднее количество ROH-сегментов и их средний суммарный размер в геноме коров росли экспоненциально. При этом средний размер ROH-сегментов в геноме коров менялся незначительно 10,4±1,2 Mb. В зависимость от количества гетерозиготных SNPs коэффициент инбридинга также рос экспоненциально. В случае отсутствия гетерозиготных SNPs в ROH-сегментах средний коэффициент инбридинга у коров в 6 стадах изменялся от 4,4±0,4 до 5,6±0,2. Предполагается, что полученные результаты обусловлены не случайным расположением ROH-сегментов, а их кластеризацией в хромосомах коров. Таким образом, для несмещенной оценки результатов ROH-анализа следует исключать гетерозиготные SNPs в ROH-сегментах. Russian Research Institute of Farm Animal Genetics and Breeding Branch of the L.K. Ernst Federal Science Center for Animal Husbandry The aim of this study was to assess the effect of non-genotyped and heterozygous SNPs in ROH segments at the average number and average total size of ROH segments, as well as at the calculation of the inbreeding coefficient of Holsteinized Back-and-White cows. Of the six breeding farms in the Leningrad Region, 374 cows were selected. Whole — genome genotyping was carried out with the BovineSNP50 v2 chip. When performing quality control of the genotyped cows and genome-wide ROH runs, the Plink 1.9 program was used. Upon quality control of the SNP genotypes out of 54609, 43298 SNPs were remained. The effect of non-genotyped and heterozygous SNPs at the results of ROH runs was evaluated by changing these parameters from 0 to 16. It turned out that non-genotyped SNPs did not affect the ROH scores, while the number of allowed heterozygous SNPs significantly affected the results of ROH runs. Thus, as the number of allowed heterozygous SNPs in ROH segments were increased, the mean number of ROH segments and their mean total size in the cows’ genome increased exponentially. Nevertheless, the mean size of the ROH segment changed slightly 10.4±1.2 Mb. Depending on the number of heterozygous SNPs in ROH, the inbreeding coefficient as well increased exponentially. In the absence of heterozygous SNPs in ROH segments, the average inbreeding coefficient of the cows in six herds varied from 4.4±0.4 to 5.6±0.2. It is suggested that obtained results are not due to the random distribution of ROH segments, but their clustering in the chromosomes of the cows. Thus, for unbiased assessment of the results of ROH analysis, heterozygous SNPs in ROH segments should be excluded.

Genomic analyses provide insights into breed-of-origin effects from purebreds on three-way crossbred pigs

Crossbreeding is widely used aimed at improving crossbred performance for poultry and livestock. Alleles that are specific to different purebreds will yield a large number of heterozygous single-nucleotide polymorphisms (SNPs) in crossbred individuals, which are supposed to have the power to alter gene function or regulate gene expression. For pork production, a classic three-way crossbreeding system of Duroc × (Landrace × Yorkshire) (DLY) is generally used to produce terminal crossbred pigs with stable and prominent performance. Nonetheless, little is known about the breed-of-origin effects from purebreds on DLY pigs. In this study, we first estimated the distribution of heterozygous SNPs in three kinds of three-way crossbred pigs via whole genome sequencing data originated from three purebreds. The result suggested that DLY is a more effective strategy for three-way crossbreeding as it could yield more stably inherited heterozygous SNPs. We then sequenced a DLY pig family and identified 95, 79, 132 and 42 allele-specific expression (ASE) genes in adipose, heart, liver and skeletal muscle, respectively. Principal component analysis and unrestricted clustering analyses revealed the tissue-specific pattern of ASE genes, indicating the potential roles of ASE genes for development of DLY pigs. In summary, our findings provided a lot of candidate SNP markers and ASE genes for DLY three-way crossbreeding system, which may be valuable for pig breeding and production in the future.

Haplotype-Contained PCR Products Analysis by Sequencing with Selective Restriction of Primer Extension

We develop a strategy for haplotype analysis of PCR products that contained two adjacent heterozygous loci using sequencing with specific primers, allele-specific primers, and ddNTP-blocked primers. To validate its feasibility, two sets of PCR products, including two adjacent heterozygous SNPs, UGT1A1⁎6 (rs4148323) and UGT1A1⁎28 (rs8175347), and two adjacent heterozygous SNPs, K1637K (rs11176013) and S1647T (rs11564148), were analyzed. Haplotypes of PCR products, including UGT1A1⁎6 and UGT1A1⁎28, were successfully analyzed by Sanger sequencing with allele-specific primers. Also, haplotypes of PCR products, including K1637K and S1647T, could not be determined by Sanger sequencing with allele-specific primers but were successfully analyzed by pyrosequencing with ddNTP-blocked primers. As a result, this method is able to effectively haplotype two adjacent heterozygous PCR products. It is simple, fast, and irrespective of short read length of pyrosequencing. Overall, we fully hope it will provide a new promising technology to identify haplotypes of conventional PCR products in clinical samples.