RESUMEN
The successful accomplishment of the first telomere-to-telomere human genome assembly, T2T-CHM13, marked a milestone in achieving completeness of the human reference genome. The upcoming era of genome study will focus on fully phased diploid genome assembly, with an emphasis on genetic differences between individual haplotypes. Most existing sequencing approaches only achieved localized haplotype phasing and relied on additional pedigree information for further whole-chromosome scale phasing. The short-read-based Strand-seq method is able to directly phase single nucleotide polymorphisms (SNPs) at whole-chromosome scale but falls short when it comes to phasing structural variations (SVs). To shed light on this issue, we developed a Nanopore sequencing platform-based Strand-seq approach, which we named NanoStrand-seq. This method allowed for de novo SNP calling with high precision (99.52%) and acheived a superior phasing accuracy (0.02% Hamming error rate) at whole-chromosome scale, a level of performance comparable to Strand-seq for haplotype phasing of the GM12878 genome. Importantly, we demonstrated that NanoStrand-seq can efficiently resolve the MHC locus, a highly polymorphic genomic region. Moreover, NanoStrand-seq enabled independent direct calling and phasing of deletions and insertions at whole-chromosome level; when applied to long genomic regions of SNP homozygosity, it outperformed the strategy that combined Strand-seq with bulk long-read sequencing. Finally, we showed that, like Strand-seq, NanoStrand-seq was also applicable to primary cultured cells. Together, here we provided a novel methodology that enabled interrogation of a full spectrum of haplotype-resolved SNPs and SVs at whole-chromosome scale, with broad applications for species with diploid or even potentially polypoid genomes.
RESUMEN
There is no effective way to detect structure variations (SVs) and extra-chromosomal circular DNAs (ecDNAs) at single-cell whole-genome level. Here, we develop a novel third-generation sequencing platform-based single-cell whole-genome sequencing (scWGS) method named SMOOTH-seq (single-molecule real-time sequencing of long fragments amplified through transposon insertion). We evaluate the method for detecting CNVs, SVs, and SNVs in human cancer cell lines and a colorectal cancer sample and show that SMOOTH-seq reliably and effectively detects SVs and ecDNAs in individual cells, but shows relatively limited accuracy in detection of CNVs and SNVs. SMOOTH-seq opens a new chapter in scWGS as it generates high fidelity reads of kilobases long.
Asunto(s)
Mapeo Cromosómico/métodos , Neoplasias Colorrectales/genética , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de la Célula Individual/métodos , Secuenciación Completa del Genoma/métodos , Secuencia de Bases , Neoplasias Colorrectales/patología , Variaciones en el Número de Copia de ADN , Elementos Transponibles de ADN , ADN Circular/genética , Células HEK293 , Humanos , Células K562 , Polimorfismo de Nucleótido SimpleRESUMEN
Chromosome translocation is a major cause of the onset and progression of diverse types of cancers. However, the mechanisms underlying this process remain poorly understood. Here, we identified a non-homologous end-joining protein, IFFO1, which structurally forms a heterotetramer with XRCC4. IFFO1 is recruited to the sites of DNA damage by XRCC4 and promotes the repair of DNA double-strand breaks in a parallel pathway with XLF. Interestingly, IFFO1 interacts with lamin A/C, forming an interior nucleoskeleton. Inactivating IFFO1 or its interaction with XRCC4 or lamin A/C leads to increases in both the mobility of broken ends and the frequency of chromosome translocation. Importantly, the destruction of this nucleoskeleton accounts for the elevated frequency of chromosome translocation in many types of cancer cells. Our results reveal that the lamin A/C-IFFO1-constituted nucleoskeleton prevents chromosome translocation by immobilizing broken DNA ends during tumorigenesis.
Asunto(s)
Carcinogénesis/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Lamina Tipo A/metabolismo , Translocación Genética , Animales , Carcinoma/genética , Cromosomas Humanos , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Proteínas de Filamentos Intermediarios/genética , Ratones , Matriz Nuclear/metabolismo , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Asociadas a Matriz Nuclear/fisiologíaRESUMEN
A reversed-phase high performance liquid chromatographic method for simultaneous determination of 18 amino acids is described. The sample was mixed with norleucine as the internal standard, then derivatized with phenylisothiocyanate (PITC) and analyzed on a Kromasil C18 column at 38 degrees C, using gradient elution with detection at 254 nm. The correlation coefficients between the ratios of peak area of amino acid to that of the internal standard and the amino acid concentrations were above 0.99, except for cysteine(Cys)(0.962). The recoveries of amino acids added were 96.0%-102.4%. Detection limit of leucine was 0.5 mg/L. The method was applied to analyze free amino acids in deproteinized calf blood injection and good results were obtained.