In 2003, history was made. For the primary time, the human genome was sequenced. Since then, technological improvements have enabled tweaks, adjustments, and additions, making the human genome the foremost accurate and complete vertebrate genome ever sequenced.
Nevertheless, some gaps remain - including human chromosomes. we have a fairly good grasp of them normally, but there are still some gaps within the sequences. Now, for the primary time, geneticists have closed a number of those gaps, giving us the primary complete, gap-free, end-to-end (or telomere-to-telomere) sequence of an individual's X chromosome.
The accomplishment was enabled by a brand new technique called nanopore sequencing, which enables ultra-long-reads of DNA strands, providing a more complete and sequential assembly.
This is in contrast to previous sequencing techniques, during which only short sections may be read at a time. Previously, geneticists had to piece together these sections sort of a puzzle.
While they were pretty good at this, the pieces tend to seem identical, so it is very tricky to grasp if you're getting it right - not just the proper order, but what percentage repeats there are within the sequence. And, of course, there are minute gaps.
"We're setting out to find that a number of these regions where there have been gaps within the reference sequence are literally among the richest for variation in human populations, so we've been missing lots of data that would be important to understanding human biology and disease," said satellite DNA biologist Karen Miga of the University of California Santa Cruz Genomics Institute.
This is where nanopore sequencing comes in. It consists of a protein nanopore - a nanoscale hole - set in an electrically resistant membrane. Current is applied to the membrane, which passes it through the nanopore. When genetic material is fed into the nanopore, the change in current may be translated into a genetic sequence.
Even better, this technology reduces reliance on polymerase chain reaction, a way that amplifies DNA by creating voluminous copies of it.
It was this system that Miga and her team wont to study DNA obtained from a rare variety of benign uterine tumors, an abnormal condition, together with other sequencing technologies - Illumina and PacBio - to create sure the tip result was as complete as possible.
"We used an iterative process over three different sequencing platforms to shine the sequence and reach a high level of accuracy," Miga said. "The unique markers provide an anchoring system for the ultra-long reads, and once you anchor the reads, you'll be able to use multiple data sets to call each base."
Even with these back-ups, though, there still remained some gaps - most notably within the centromere, the structure that connects the chromatids: thread-like strands into which a chromosome divides. This region is significant for mitosis - but it is also very complex. within the X chromosome, it is a highly repetitive region spanning 3.1 million DNA base pairs.
The researchers were able to resolve this notoriously tricky structure by trying to find slight variations within the repeats. These variations allowed the scientists to align and connect the long reads to make a whole sequence for the centromere.
"For me, the thought that we will put together a 3-megabase-size tandem repeat is simply mind-blowing," Miga said. "We can now reach these repeat regions covering countless bases that were previously thought intractable."
This rigorous approach allowed the team to shut all 29 gaps within the current sex chromosome reference. it is a major breakthrough within the project to completely map the human genome.
"Our results demonstrate that finishing the whole human genome is now reachable," the researchers wrote in their paper, "and the info presented here will enable ongoing efforts to complete the remaining human chromosomes."
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