But after the project claimed victory in 2003, Eichler was only a little closer to his scientific goal. The sequencing effort had failed to read many big chunks of DNA \u2013 more than eight percent of the genome. Scientists knew these missing chunks contained highly repetitive sequences, and largely dismissed them as junk. Not so, says Eichler, a Howard Hughes Medical Institute (HHMI) Investigator at the University of Washington. \u201cIt turned out that many of the regions I was interested in were in the gaps.\u201d He became committed to finishing the job \u2013 reading the entire genome, tricky bits and all.<\/p>\n
Now he and a team of about 100 scientists, led by Adam Phillippy of the National Human Genome Research Institute (NHGRI) and Karen Miga of the University of California, Santa Cruz, (UCSC) have finally gotten it right. In new work\u00a0first posted as a preprint on bioRxiv.org<\/a>\u00a0and now published March 31, 2022, in the journal\u00a0Science<\/em>, they describe the\u00a0first ever sequencing of an entire human genome<\/a>, adding a whole chromosome\u2019s worth of previously hidden DNA \u2013 the missing eight percent. In the genetic manuscript for life, \u201cwe are seeing chapters that were never read before,\u201d says Eichler.<\/p>\n Or as University of Washington geneticist Robert Waterston puts it: \u201cThere are no longer any hidden or unknown bits.\u201d<\/p>\n \u201cI think that is psychologically a big thing,\u201d adds Waterson, a leader in the original Human Genome Project who was not involved in the new effort. \u201cI just admire these scientists for sticking with it.\u201d<\/p>\n An intricate puzzle\u00a0<\/strong><\/p>\n The human genome is made up of just over six billion individual letters of DNA \u2013 about the same number as other primates like chimps \u2013 spread among 23 pairs of chromosomes. To read a genome, scientists first chop up all that DNA into pieces hundreds to thousands of letters long. Sequencing machines then read the individual letters in each piece, and scientists try to assemble the pieces in the right order, like putting together an intricate puzzle.<\/p>\n One challenge is that some regions of the genome repeat the same letters over and over again. Repetitive regions include the centromeres, the parts that hold the two strands of chromosomes together and that play crucial roles in cell division, and ribosomal DNA, which provides instructions for the cell\u2019s protein factories. Still other repetitive parts include new genes that may help species adapt. In the past, all that repetition made it impossible to assemble some chopped-up pieces in the correct order. It\u2019s like having identical puzzle pieces \u2013 scientists didn\u2019t know which went where, leaving big gaps in the genomic picture.<\/p>\n Another snag: most cells contain two genomes \u2013 one from the father and one from the mother. When researchers try to assemble all the pieces, sequences from each parent can mix together, obscuring the actual variation within each individual genome.<\/p>\n In the mid-2000s, as scientists tried to figure out how to overcome the barriers, \u201cwe came up with the idea of getting a complete genome by sequencing just one of the genomes instead of solving two at the same time,\u201d recalls Eichler. He knew just where to find it \u2013 from a set of cell lines being studied by University of Pittsburgh reproductive geneticist Urvashi Surti. Because of a rare glitch in normal development, the cells end up with two copies of the father\u2019s DNA and none of the mother\u2019s.<\/p>\n Such a cell line, with only one genome, \u201cis what made this genome assembly possible,\u201d says HHMI Investigator\u00a0Erich Jarvis<\/a>, a Rockefeller University neurogeneticist who collaborated on the new work.<\/p>\n Fired up<\/strong><\/p>\n Other key advances included rapid improvements in the gene sequencing machines made by Oxford Nanopore Technologies and Pacific Biosciences. By 2017, NHGRI\u2019s Phillippy and UCSC\u2019s Miga realized that a new Nanopore machine\u2019s ability to accurately read a million letters of DNA at a time had opened the door to finally tackling the genome\u2019s hard bits. They created the Telomere-to-Telomere (T2T) consortium to sequence each chromosome from one end, or telomere, to the other. Around the same time Eichler\u2019s team had shown the value of using Pacific Biosciences technology to resolve more complex forms of genetic variation.<\/p>\n There was no guarantee of success. But \u201cwe had the benefit of youthful optimism and we were fired up by the promise of these new technologies,\u201d recalls Phillippy. The team ran their Nanopore machines nonstop for six months and brought in scores of scientists to assemble the pieces and analyze the results. At the same time, sequencing data were being generated by other team members and Pacific Biosciences using their long-read sequencing platform.\u00a0 In particular, the project got a boost when Pacific Biosciences introduced a new sequencing machine which generated long-read sequencing reads that were greater than 99 percent accurate. \u201cIt was the last piece of the puzzle \u2013 like putting on a new pair of glasses,\u201d says Phillippy. The Pacific Biosciences technology couldn\u2019t cover all parts of the genome equally well, but the scientists realized that by combining the long-read sequencing with the Oxford Nanopore data, they could fill all the gaps.<\/p>\n By summer 2020, the consortium had assembled two chromosomes and planned what Phillippy calls a hackathon to get the other 21, working remotely over Zoom and Slack during the pandemic. One key aha moment came when the team tried to assemble the most difficult regions of the genome \u2013 the highly repetitive DNA in the centromeres. The researchers realized that the algorithms for assembling the pieces couldn\u2019t handle the repetition, but the human eye could. On the computer screen, the scientists saw where the different repetitive sequences had become tangled together. Then, they untangled it manually, \u201clike untangling a string in your yo-yo,\u201d Jarvis says. By summer\u2019s end, the team had sequenced every chromosome.<\/p>\n![\"\"](\"https:\/\/www.fie.undef.edu.ar\/ceptm\/wp-content\/uploads\/2022\/04\/eichler-evan-1500x1000px.jpg\")
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