Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The process of reprogramming and recloning.

<p>(A) Phase contrast light microscopic observation of cells during reprogramming and recloning. Images captured on a Pronectin F-coated dish prior to colony picking on days four, nine, 13, 17 and 24 (upper panels). Note that human ES cell-like colonies emerged within a cobblestone like morphology. (B) Efficiency of generating reprogrammed cells on various coating materials and the number of colonies characterized. All the experiments used 1×10⁴ CD34⁺ CBCs, SeV TS vectors at 20 M.O.I. and ReproFF medium. Three independent experiments for Matrigel, Pronectin F and two independent experiments for fibronectin and laminin (Laminin-extracts), Pro<i>nectin L we</i>re performed. (C) Frequency of generating human ES cell-like colonies in various culture media. Five thousand CD34⁺ CBCs were infected with 20 M.O.I. of SeV carrying four factors and cultured in ReproFF, ReproFF2, mTeSR1, or E8 medium on Pronectin F-coated dishes to reprogram CD34+ CBCs. D: Endogenous gene expression of <i>Klf4, c-Myc, Oct3/4,</i> and <i>Sox2</i> in feeder like-cells (lane 1) and first pick up of a human ES cell like-colony (lane 2).</p

Expression of surface molecules on CD34⁺ cells and iPSCs.

<p>(A) Adhesion molecules integrin α5, β1, syndecan-2, and -4 on CD34⁺ CBCs (upper panels) and iPSC colonies (lower panels) detected by immunostaining with the relevant antibody. Alexa 594- and Alexa 488-conjugated secondary antibodies (red and green, respectively) were used to visualize the staining. Nuclei were stained with DAPI (lower photos). Means of the percentages of positive cells with standard deviation are appended in the right top of the photos. (B) Protocol for generation of iPSCs from CD34⁺ CBCs on Pronectin F-coated dishes with temperature sensitive SeV vectors. P: passage.</p

Generation of reprogrammed cell clone from a single cell via the naïve state.

<p>Human ES cell-like colonies (first prime state) were picked up at day 24 and cultured on Pronectin F-coated dishes. The colonies were subjected to heat treatment (38°C, three days) at passage three (P3). Light microscopic image and ALP staining at P3 are shown in upper and lower panels, respectively. Colonies emerged from single cells in Pronectin F-coated 96-well plates under naïve conditions at P4, dome-shaped colonies at P5 under naïve conditions, ES cell-like colonies (second primed) cultured under primed culture conditions at P6 or long-term passaged clone (PFX#9) at P45 are shown.</p