Nanopore sequencing and assembly of a human genome with ultra-long reads

We report the sequencing and assembly of a reference genome for the human GM12878 Utah/Ceph cell line using the MinION (Oxford Nanopore Technologies) nanopore sequencer. 91.2 Gb of sequence data, representing ~30× theoretical coverage, were produced. Reference-based alignment enabled detection of large structural variants and epigenetic modifications. De novo assembly of nanopore reads alone yielded a contiguous assembly (NG50 ~3 Mb). Next, we developed a protocol to generate ultra-long reads (N50 > 100kb, up to 882 kb). Incorporating an additional 5×-coverage of these data more than doubled the assembly contiguity (NG50 ~6.4 Mb). The final assembled genome was 2,867 million bases in size, covering 85.8% of the reference. Assembly accuracy, after incorporating complementary short-read sequencing data, exceeded 99.8%. Ultra-long reads enabled assembly and phasing of the 4 Mb major histocompatibility complex (MHC) locus in its entirety, measurement of telomere repeat length and closure of gaps in the reference human genome assembly GRCh38

Persistent activation of NF-kB in cultured rat hepatic stellate cells involves the induction of potentially novel rel-like factors and prolonged changes in the expression of IkB family proteins.

Rat hepatic stellate cells (HSC) cultured in serum-containing medium underwent a rapid (3-hour) classical induction of p50:p65 and p65:p65 nuclear factor-kappa B (NF-kappa B) dimers, Subsequent culturing was associated with prolonged expression of active p50:p65 and persistent induction of a high-mobility NF-kappa B DNA binding complex consisting of potentially novel Rel-like protein(s). Formation of the latter complex was competed for by specific double-stranded oligonucleotides, was up-regulated by treatment of HSCs with tumor necrosis factor alpha (TNF-alpha), and was maintained at basal levels of expression by a soluble HSC-derived factor. An NF-kappa B-responsive CAT reporter gene was highly active in early cultured HSCs but was also trans-activated at a lower but significant level in longer-term cultured cells and could be completely suppressed by expression of dominant negative I kappa B-alpha. Physiological significance of the lower persistent NF-kappa B activities was also demonstrated by the ability of long-term cultured HSCs to support the activity of the NF-kappa B-dependent human intercellular adhesion molecule-1 (ICAM-1) promoter. Freshly isolated HSCs expressed high levels of I kappa B-alpha and I kappa B-beta. Culture activation was accompanied by a long-term reduction in levels of I kappa B-alpha With no detectable expression in the nuclear fraction of cells, under these conditions p50:p65 was detected in the nucleus. I kappa B-beta expression was transiently reduced and, upon replenishment, was associated with appearance of a lower-mobility I kappa B-beta antibody-reactive species. Bcl3 expression was absent in freshly isolated HSC but was induced during culturing and became a persistent feature of the activated HSC, Inhibition of NF-kappa B DNA binding activity by gliotoxin was associated with increased numbers of apoptotic cells. We suggest that activation of NF-kappa B in cultured HSC is required for expression of specific genes associated with the activated phenotype such as ICAM-1 and may be antiapoptotic for rat HSCs.</p

Characterisation of hepatic fibrosis by genetic profiling of in vitro activated human stellate cells

Hepatic fibrosis is a progressive debilitating disease which if left unchecked leads to cirrhosis and liver failure. To date, transplantation remains the only clinical treatment. The molecular mechanisms which lead to fibrosis represent an area of intensive study and it is now clear that hepatic stellate cells (HSCs) play a pivotal role in this process. As a first step to developing an effective anti-fibrotic chemotherapy, we have undertaken a global genetic analysis of HSC activation using Affymetrix GeneChip™ HG-U95A, which represents up to 11 000 characterised human genes.We have analysed pairwise, matched HSCs either freshly isolated (designated as “quiescent”), or activated by culture on plastic in the presence of 16% serum, from three independent healthy human liver samples at n=&gt;4 for each condition. All three datasets show uniform host control gene expression and hybridisation controls. Our three datasets exhibit a consistent number of expressed genes (4000–5000 active in both quiescent and activated HSCs). In addition, ~2200 genes are consistently modulated upon activation, of which ~1000 genes are upregulated and ~1200 are downregulated. Furthermore, 900 gene modulations (~40%) are common to all three samples which is extremely high agreement when compared to other gene profiling studies using established or transformed cell lines.Finally, further analysis shows that ~400 genes are consistently and significantly upregulated during HSC activation. Many of the collagen genes are represented in the upregulated genes as expected for a model for fibrosis. Other identified genes which are modulated include those for cell motility, growth factors/receptors, TGF signalling, ECM remodelling, and those genes which are established markers for activation—for example, ?-smooth muscle actin.We intend using these datasets to target pathways for therapeutic intervention to help prevent hepatic fibrosis caused, for example by chronic HCV infection. Such an anti-fibrotic therapy, allied to an anti-HCV viral therapy, would meet an urgent clinical nee