Carryover Contamination-Controlled Amplicon Sequencing Workflow for Accurate Qualitative and Quantitative Detection of Pathogens: a Case Study on SARS-CoV-2.

Carryover contamination during amplicon sequencing workflow (AMP-Seq) put the accuracy of the high-throughput detection for pathogens at risk. The purpose of this study is to develop a carryover contaminations-controlled AMP-Seq (ccAMP-Seq) workflow to enable accurate qualitative and quantitative detection for pathogens. By using the AMP-Seq workflow to detect SARS-CoV-2, Aerosols, reagents and pipettes were identified as potential sources of contaminations and ccAMP-Seq was then developed. ccAMP-Seq used filter tips and physically isolation of experimental steps to avoid cross contamination, synthetic DNA spike-ins to compete with contaminations and quantify SARS-CoV-2, dUTP/uracil DNA glycosylase system to digest the carryover contaminations, and a new data analysis procedure to remove the sequencing reads from contaminations. Compared to AMP-Seq, the contamination level of ccAMP-Seq was at least 22-folds lower and the detection limit was also about an order of magnitude lower-as low as one copy/reaction. By testing the dilution series of SARS-CoV-2 nucleic acid standard, ccAMP-Seq showed 100% sensitivity and specificity. The high sensitivity of ccAMP-Seq was further confirmed by the detection of SARS-CoV-2 from 62 clinical samples. The consistency between qPCR and ccAMP-Seq was 100% for all the 53 qPCR-positive clinical samples. Seven qPCR-negative clinical samples were found to be positive by ccAMP-Seq, which was confirmed by extra qPCR tests on subsequent samples from the same patients. This study presents a carryover contamination-controlled, accurate qualitative and quantitative amplicon sequencing workflow that addresses the critical problem of pathogen detection for infectious diseases. IMPORTANCE Accuracy, a key indicator of pathogen detection technology, is compromised by carryover contamination in the amplicon sequencing workflow. Taking the detection of SARS-CoV-2 as case, this study presents a new carryover contamination-controlled amplicon sequencing workflow. The new workflow significantly reduces the degree of contamination in the workflow, thereby significantly improving the accuracy and sensitivity of the SARS-CoV-2 detection and empowering the ability of quantitative detection. More importantly, the use of the new workflow is simple and economical. Therefore, the results of this study can be easily applied to other microorganism, which has great significance for improving the detection level of microorganism.

Diagnosis of prenatal 22q11.2 duplication syndrome: a two-case study.

The objective of the study was to perform the prenatal diagnosis of two foetuses with 22q11.2 duplication for 2.5 Mb after noninvasive prenatal testing (NIPT), and to explore the prenatal diagnosis and genetic characteristics of these foetuses. After amniocentesis, each foetus was diagnosed through karyotype analysis and single-nucleotide polymorphism array (SNP-array), and copy number variation using shotgun sequencing (CNV-seq) was carried out on each mother's peripheral blood for comparative analysis. Both pregnant woman 1 and pregnant woman 2 had foetal amniotic fluid chromosomal karyotypes of 46, XN. The SNP-array result for foetus 1 was arr[hg19] 22q11.21(18,648,856-21,800,471) x3; namely, 22q11.2 had a 3.1 Mb repeat, and the SNP-array result of foetus 2 was arr[hg19]22q11.2(18,648,855-21,464,764) x3; there was a 2.4 Mb repeat of 22q11.2. The CNV-Seq result of the peripheral blood of pregnant woman 1 was seq[hg19]22q11.2(18,953,139-21,449,967) x3; namely, in this mother's 22q11.2 region, there was ~2.5 Mb of duplicate fragment that was pathogenic to CNV. We confirmed that case 1 was inherited from the mother by CNV-seq. In both cases, however, there were key region deletions, including 41 OMIM genes such as CLTCL1, HIRA and TBX1. Both SNP-array and CNV-seq can effectively diagnose 22q11.2 duplication syndrome and clarify its fracture site and involved genes, which may facilitate understanding of the genotype and phenotype correlations.

Regulation of inflorescence branch development in rice through a novel pathway involving the pentatricopeptide repeat protein sped1-D.

Panicle type has a direct bearing on rice yield. Here, we characterized a rice clustered-spikelet mutant, sped1-D, with shortened pedicels and/or secondary branches, which exhibits decreased pollen fertility. We cloned sped1-D and found that it encodes a pentatricopeptide repeat protein. We investigated the global expression profiles of wild-type, 9311, and sped1-D plants using Illumina RNA sequencing. The expression of several GID1L2 family members was downregulated in the sped1-D mutant, suggesting that the gibberellin (GA) pathway is involved in the elongation of pedicels and/or secondary branches. When we overexpressed one GID1L2, AK070299, in sped1-D plants, the panicle phenotype was restored to varying degrees. In addition, we analyzed the expression of genes that function in floral meristems and found that RFL and WOX3 were severely downregulated in sped1-D. These results suggest that sped1-D may prompt the shortening of pedicels and secondary branches by blocking the action of GID1L2, RFL, and Wox3. Moreover, overexpression of sped1-D in Arabidopsis resulted in the shortening of pedicels and clusters of siliques, which indicates that the function of sped1-D is highly conserved in monocotyledonous and dicotyledonous plants. Sequence data from this article have been deposited with the miRBase Data Libraries under accession no. MI0003201.

[Molecular mapping of a bacterial blight resistance gene Xa-25 in rice].

Xa-25 was a bacterial blight resistance gene identified in a somaclonal mutant HX-3. A doubled-haploid (DH) population including 129 stable lines was derived from anther culture of a typical japanica 02428 and a typical indica HX-3 cross. The bacterial blight strain Zhe173, a typical bacterial blight strain in Yangtze River valley, was used to test the resistance or susceptible of the DH population lines, and the results showed that the resistance lines and susceptible lines were 62 and 67, respectively. A total of 300 SSR primer pairs covering 12 rice chromosomes were used for polymorphism survey of 02428 and HX-3. Among these primers, 74 showed polymorphism between the parents. Using these polymorphic SSR markers, bulked segregant analysis was conducted on the DH population. As the result, Xa-25 was located at the terminal region of the long arm of chromosome 4 between the two SSR markers RM6748 and RM1153, the map distance between Xa-25 and the two SSR markers was 9.3 cM and 3.0 cM, respectively.