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

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