Biology subjects

Greer, S. U.

Publications and source records attributed to Greer, S. U..

8 recordsLinked to original sources

bioRxiv2022

The Human Pangenome's sequence conservation reveals a landscape of polymorphic structural variation

The Human Pangenome is a new reference build that addresses many of the limitations of the current reference. The first release is based on 94 high-quality haploid assemblies from individuals with diverse backgrounds. To facilitate the Pangenomes adoption in the wider research community, we conducted a multiple genome comparative analysis against the current GRCh38 reference. We applied a k-mer indexing strategy to identify highly conserved sequences that are omnipresent across all Pangenome assemblies, the reference and CHM13, a reference assembly with telomere-to-telomere chromosomes. Pan-conserved tag segments provided an informative set of universally conserved sequences. Examining the intervals between pairs of these segments defined highly conserved segments of the genome versus ones that have structurally related polymorphisms. We identified a Pangenome landscape of 60,764 polymorphic intervals with unique and geo-ethnic features. Overall, this study of the Pangenome revealed the conserved versus divergent features including the landscape of polymorphic structural variants.

genomics↗

bioRxiv2021

Characterization of the consensus mucosal microbiome of colorectal cancer

Recent evidence suggests that dysbiosis, an imbalance of microbiota, is associated with increased risk of colorectal cancer. Diverse microbial organisms are physically associated with the cells found in tumor biopsies. Characterizing this mucosa-associated microbiome through genome sequencing has advantages compared to culture-based profiling. However, there are notable challenges in accurately characterizing the features of tumor microbiomes with methods like transcriptome sequencing. Most sequence reads originate from the host. Moreover, there is a high likelihood of bacterial contaminants being introduced. Another major challenge is the microbiome diversity among different studies. Colorectal tumors demonstrate a significant extent of microbiome variation among individuals from different geographic and ethnic origins. To address these challenges, we identified a consensus microbiome for colorectal cancer through analyzing 924 tumors from eight independent RNA-Seq data sets. A standardized meta-transcriptomic analysis pipeline was established and applied to the complete CRC cohort. Common contaminants were filtered out. Our study involved taxonomic investigation of non-human sequences, linked microbial signatures to phenotypes and the association of microbiome with tumor microenvironment components. Microbiome profiles across different CRC cohorts were compared, and recurrently altered microbial shifts specific to CRC were determined. We identified cancer-specific set of 114 microbial species associated with tumors that were found among all investigated studies. Validating our approach, we found that Fusobacterium nucleatum was one of the most enriched bacterial species in CRC. Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria were among the four most abundant phyla for CRC microbiome. Signficant associations between the consensus species and specific immune cell types were noted. Our results are available as a web data resource for other researchers to explore (https://crc-microbiome.stanford.edu).

genomics↗

bioRxiv2021

KmerKeys: a web resource for searching indexed genome assemblies and variants

K-mers are short DNA sequences that are used for genome sequence analysis. Applications that use k-mers include genome assembly and alignment. Despite these current applications, the wider bioinformatic use of k-mers in has challenges related to the massive scale of genomic sequence data. A single human genome assembly has billions of these short sequences. The sheer amount of computation for effective use of k-mer information is enormous, particularly when involving multiple genome assemblies. To address these issues, we developed a new k-mer indexing data structure based on a hash table tuned for the lookup of k-mer keys. This web application, referred to as KmerKeys (https://kmerkeys.dgi-stanford.org/), provides performant, rapid query speeds for cloud computation on genome assemblies. We enable fuzzy as well as exact k-mer-based searches of assemblies. To enable robust and speedy performance, the website implements cache-friendly hash tables, memory mapping and massive parallel processing. Our method employs a scalable and efficient data structure that can be used to jointly index and search a large collection of human genome assembly information. One can include variant databases and their associated metadata such as the gnomAD population variant catalog. This feature enables the incorporation of future genomic information into sequencing analysis.

bioinformatics↗

bioRxiv2020

Profiling diverse sequence tandem repeats in colorectal cancer reveals co-occurrence of microsatellite and chromosomal instability involving Chromosome 8

Colorectal carcinomas (CRCs) which have lost DNA mismatch repair display hypermutability evident in a molecular phenotype called microsatellite instability (MSI). These mismatch repair deficient tumors are thought to lack widespread genomic instability features, such as copy number changes and rearrangements. To identify MSI for clinical diagnosis, current molecular testing looks for changes in mononucleotide or dinucleotide repeats. However, microsatellites have other types of sequence tandem repeats such as tri- and tetranucleotide motifs. These additional classes of microsatellites are generally not examined for MSI but are known to be unstable in a phenotype known as elevated microsatellite alterations at selected tetranucleotide repeats, or EMAST. We developed a sequencing approach that provides ultra-high coverage (>2500X) of microsatellite targets and cancer genes for profiling genomic instability. We assessed the diverse repeat motifs across 200 microsatellites. Our approach provides highly sensitive detection of MSI with high specificity, evaluates copy number alterations with high accuracy, delineates chromosomal instability (CIN) classification and deconvolutes subclonal architecture. By examining both MSI and CIN, we discovered mutations and copy number alterations that defined mixed genomic instability states of CIN and MSI, which are normally considered exclusive. An increase in copy number of chromosome arm 8q was prevalent among MSI tumors. Moreover, we identified an inter-chromosomal translocation event from a CRC with co-occurrence of MSI. Subclonal analysis demonstrated that mutations which are typically considered to be exclusive in MSI, shows mutual occurrence in MSI tumors with more sensitive characterization. Our approach revealed that MSH3 mutations are a potential source of mixed genomic instability features. Overall, our study demonstrates that some colorectal cancers have features of both microsatellite and chromosomal instability. This result may have implications for immunotherapy treatment.

genomics↗

bioRxiv2020

Complete and haplotype-specific sequence assembly of segmental duplication-mediated genome rearrangements using targeted CRISPR-targeted ultra-long read sequencing (CTLR-Seq)

We have developed a generally applicable method based on CRISPR/Cas9-targeted ultra-long read sequencing (CTLR-Seq) to completely and haplotype-specifically resolve, at base-pair resolution, large, complex, and highly repetitive genomic regions that had been previously impenetrable to next-generation sequencing analysis such as large segmental duplication (SegDup) regions and their associated genome rearrangements that stretch hundreds of kilobases. Our method combines in vitro Cas9-mediated cutting of the genome and pulse-field gel electrophoresis to haplotype-specifically isolate intact large (200-550 kb) target regions that encompass previously unresolvable genomic sequences. These target fragments are then sequenced (amplification-free) to produce ultra-long reads at up to 40x on-target coverage using Oxford nanopore technology, allowing for the complete assembly of the complex genomic regions of interest at single base-pair resolution. We applied CTLR-Seq to resolve the exact sequence of SegDup rearrangements that constitute the boundary regions of the 22q11.2 deletion CNV and of the 16p11.2 deletion and duplication CNVs. These CNVs are among the strongest known risk factors for schizophrenia and autism. We then perform de novo assembly to resolve, for the first time, at single base-pair resolution, the sequence rearrangements of the 22q11.2 and 16p11.2 CNVs, mapping out exactly the genes and non-coding regions that are affected by the CNV for different carriers.

genomics↗

bioRxiv2019

Genetic risk of cholangiocarcinoma is linked to the autophagy gene ATG7

Cholangiocarcinoma is an aggressive cancer originating from the bile duct. Although cholangiocarcinoma does occur in families, to date no specific causative gene has been identified. We identified ATG7 as a cancer susceptibility gene using a joint genetic analysis of an extended pedigree with familial cholangiocarcinoma in combination with a population genetic association study. Affected family members had a germline mutation (c.2000C>T [p.Arg659*]) in the autophagy related gene, ATG7, and all of the affected individuals had cholangiocarcinoma tumors harboring somatic genomic deletions of ATG7. From a population genetic study, we identified a germline polymorphism of ATG7 (c.1591C>G [p.Asp522Glu]) associated with increased risk of cholangiocarcinoma. The autophagy substrate p62 demonstrated a higher accumulation in tumors of p.Asp522Glu carriers compared with non-carriers indicating defective autophagy. To determine whether the germline ATG7 mutation had functional consequences, we developed an ATG7-deficient cholangiocyte cell line, derived from human bile duct, to test for autophagy-mediated lipidation activity. The germline mutation from the familial cholangiocarcinoma demonstrated a lack of lipidation activity compared to the wildtype ATG7. Moreover, in zebrafish embryos depleted of atg7, a reproducible necrotic head phenotype was rescued by injection of wildtype ATG7 but not mutant ATG7. Our findings point to ATG7 as a causative genetic risk factor for cholangiocarcinoma and implicate autophagy as a novel cancer driver mechanism.

genomics↗

bioRxiv2018

Assembly of Mb-size genome segments from linked read sequencing of CRISPR DNA targets

We developed a targeted sequencing method for intact high molecular weight (HMW) DNA targets as large as 0.2 Mb. This process uses HMW DNA isolated from intact cells, custom designed Cas9-guide RNA complexes to generate 0.1 - 0.2 Mb DNA targets, electrophoretic isolation of the DNA targets and sequencing with barcode linked reads. We used alignment methods as well as local assembly of the target regions to identify haplotypes and structural variants (SVs) across multi-Megabase genomic regions. To demonstrate the performance of this approach, we designed three assays that covered a 0.2 Mb region surrounding the BRCA1 gene, a set of 40 overlapping 0.2 Mb targets covering the entire 4-Mb MHC locus, and 18 well-characterized structural variants. Using the highly characterized NA12878 genome, we achieved on-target coverage of more than 50X, while overall whole genome coverage was approximately 4X. We generated haplotypes that completely covered each targeted locus, with a maximum size of 4 Mb (for the MHC region). This method detected structural variants such as deletions and inversions with determination of the exact breakpoints and genotypes. Even breakpoints inside highly homologous segmental duplications are precisely determined with our high-quality assemblies. Overall, this is a new method to sequence large DNA segments.

genomics↗

bioRxiv2018

Haplotype-resolved and integrated genome analysis of ENCODE cell line HepG2

The HepG2 cancer cell line is one of the most widely-used biomedical research and one of the main cell lines of ENCODE. Vast numbers of functional genomics and epigenomics datasets have been produced to characterize its biology. However, the correct interpretation such data requires an understanding of the cell lines genome sequence and genome structure. Using a variety of sequencing and analysis methods, we identified a wide spectrum of HepG2 genome characteristics: copy numbers of chromosomal segments, SNVs and Indels (corrected for aneuploidy), phased haplotypes extending to entire chromosome arms, loss of heterozygosity, retrotransposon insertions, structural variants (SVs) including complex and somatic genomic rearrangements. We also identified allele-specific expression and DNA methylation genome-wide and assembled an allele-specific CRISPR/Cas9 targeting map.\n\nSIGNIFICANCEHaplotype-resolved and comprehensive whole-genome analysis of a widely-used cell line for cancer research and ENCODE, HepG2, serves as an essential resource for unlocking complex cancer gene regulation using a genome-integrated framework and also provides genomic context for the analysis of ~1,000 functional datasets to date on ENCODE for biological discovery. We also demonstrate how deeper insights into genomic regulatory complexity are gained by adopting a genome-integrated framework.

genomics↗