THE human genome

DNA sequencing by the Sanger method

What is a genomeTypes of genomesWhat is genomicsHow is genomics different from geneticsTypes of genomicsGenome sequencingMilestones in genomic sequencingTechnical foundations of genomicsSteps of genome sequencingDNA sequencing approachesHierarchical shotgun sequencingMarkers used in mapping large genomesWhole genome shotgun sequencingNew technologiesGenome sequencing achievment in BangladeshBenefits of Genome ResearchAt a glance

WHAT IS A GENOME? Genome: One complete set of genetic information (total amount of DNA) from a haploid set of chromosomes of a single cell in eukaryotes, in a single chromosome in bacteria, or in the DNA or RNA of viruses. Basic set of chromosome in a organism. The whole hereditary information of an organism that is encoded in the DNAIn cytogenetic genome means a single set of chromosomes. It is denoted by x. Genome depends on the number of ploidy of organism.In Drosophila melanogaster (2n = 2x = 8); genome x = 4.In hexaploid Triticum aestivum (2n = 6x = 42); genome x = 7.Continue

The genome is found inside every cell, and in those that have nucleus, the genome is situated inside the nucleus. Specifically, it is all the DNA in an organelle.

The term genome was introduced by H. Winkler in 1920 to denote the complete set of chromosomal and extra chromosomal genes present in an organism, including a virus.

How many types of genomes are:Prokaryotic GenomesEukaryotic Genomes Nuclear GenomesMitochondrial GenomesCholoroplast GenomesIf not specified, genome usually refers to the nuclear genome.

Genomics is the study of the structure and function of whole genomes.Genomics is the comprehensive study of whole sets of genes and their interactions rather than single genes or proteins.According to T.H. Roderick, genomics is the mapping and sequencing to analyze the structure and organization of genome.

WHAT IS GENOMICS?

Origin of terminologyThe term genome was used by German botanist Hans Winker in 1920 Collection of genes in haploid set of chromosomesNow it encompasses all DNA in a cellGenomics is the sub discipline of molecular genetics devoted to thethe structure and function of entire genomesmapping, sequencing ,and analyzing the functions of entire genomes

The field includes studies of intro-genomic phenomena such as heterosis, epistasis, pleiotropy and other interactions between loci and alleles within the genome.

The sequence information of the genome will show; The position of every gene along the chromosome, The regulatory regions that flank each gene, and The coding sequence that determines the protein produce by each gene. How is Genomics different from Genetics?Genetics as the study of inheritance and genomics as the study of genomes.Genetics looks at single genes, one at a time, like a picture or snapshot.Genomics looks at the big picture and examines all the genes as an entire system.

Types of Genomics1. Structural: It deals with the determination of the complete sequence of genomes and gene map.This has progressed in steps as follows: (i) construction of high resolution genetic and physical maps, (ii) sequencing of the genome, and (iii) determination of complete set of proteins in an organism.2. Functional: It refers to the study of functioning of genes and their regulation and products(metabolic pathways), i.e., the gene expression patterns in organism.3. Comparative: It compare genes from different genomes to elucidate functional and evolutional relationship.

Genome sequencing is the technique that allows researchers to read the genetic information found in the DNA of anything from bacteria to plants to animals. Sequencing involves determining the order of bases, the nucleotide subunits- adenine(A), guanine(G), cytosine(C) and thymine(T), found in DNA.

Genome sequencing is figuring out the order of DNA nucleotides.

Genome SequencingChallenges of genome sequencingData produce in form of short reads, which have to be assembled correctly in large contigs and chromosomes.Short reads produced have low quality bases and vector/adaptor contaminations.Several genome assemblers are available but we have to check the performance of them to search for best one.

Milestones in Genomic Sequencing

1977; Fred Sanger; fX 174 bacteriophage (first sequenced genome ); 5,375 bpAmino acid sequence of phage proteinsOverlapping genes only in viruses

Fig: The genetic map of phage fX174 (Overlapping reading frames)

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1995; Craig Venter & Hamilton Smith;Haemophilus influenzae (1,830,137 bp) (1st free living).Mycoplasma genitalium (smallest free-living, 580,000 bp; 470 genes)1996; Saccharomyces cerevisiae; (1st eukaryote) 12,068,000 bp1997; Escherichia coli; 4,639,221 bp; Genetically more important.1999; Human chromosome 22; 53,000,000 bp2000; Drosophila melanogaster; 180,000,000 bp2001; Human; Working draft; 3,200,000,000 bp2002; Plasmodium falciparum; 23,000,000 bpAnopheles gambiea; 278,000,000 bp Mus musculus; 2,500,000,000 bp2003; Human; finished sequence, 3,200,000,000 bp2005; Oryza sativa (first cereal grain); 489,000,000 bp2006; Populus trichocarpa (first tree) ; 485,000,000 bp

Technical foundations of genomics Molecular biology: Almost all of the underlying techniques of genomics originated with recombinant-DNA technology. DNA sequencing: In particular, almost all DNA sequencing is still performed using the approach pioneered by Sanger.Library construction: Also essential to high-throughput sequencing is the ability to generate libraries of genomic clones and then cut portions of these clones and introduce them into other vectors. PCR amplification: The use of the polymerase chain reaction (PCR) to amplify DNA, developed in the 1980s, is another technique at the core of genomics approaches.

Log MWDistance

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Hybridization techniques: Finally, the use of hybridization of one nucleic acid to another in order to detect and quantitate DNA and RNA (Southern blotting). This method remains the basis for genomics techniques such as microarrays.

Break genome into smaller fragmentsSequence those smaller piecesPiece the sequences of the short fragments together

Two different methods used1. Hierarchical shotgun sequencing-Useful for sequencing genomes of higher vertebrates that contain repetitive sequences2. Whole genome Shotgun Sequencing-Useful for smaller genomesSteps of genome sequencingDNA sequencing approaches

The method preferred by the Human Genome Project is thehierarchical shotgun sequencingmethod. Also known as The Clone-by-Clone Strategythe map-based methodmap first, sequence later top-down sequencing

Hierarchical Shotgun SequencingHuman Genome Project adopted a map-based strategyStart with well-defined physical mapProduce shortest tiling path for large-insert clonesAssemble the sequence for each cloneThen assemble the entire sequence, based on the physical map

Markers for regions of the genomes are identified. The genome is split into larger fragments (50-200kb) using restriction/cutting enzymes that contain a known marker.These fragments are cloned in bacteria (E. coli) using BACs (Bacterial Artificial Chromosomes), where they are replicated and stored. The BAC inserts are isolated and the whole genome is mapped by finding markers regularly spaced along each chromosome to determine the order of each cloned.The fragments contained in these clones have different ends, and with enough coverage finding ascaffoldofBAC contigs. This scaffold is called atiling path. BAC contig that covers the entire genomic area of interest makes up the tiling path. Each BAC fragment in the Golden Path is fragmented randomly into smaller pieces and these fragments are individually sequenced using automated Sanger sequencing and sequenced on both strands.These sequences are aligned so that identical sequences are overlapping. Assembly of the genome is done on the basis of prior knowledge of the markers used to localize sequenced fragments to their genomic location. A computer stitches the sequences up using the markers as a reference guide.In The Clone-by-Clone StrategyContinue

Fig: Hierarchical shotgun sequencingIn this approach, every part of the genome is actually sequenced roughly 4-5 times to ensure that no part of the genome is left out.

The Clone-by-Clone Strategy used in S. cerevisiae (yeast), C. elegans (nematode), Arabidopsis thaliana (mustard weed), Oryza sativa, Drosophila melanogaster and Homo sapiens (Human), etc.Each 150,000 bp fragment is inserted into a BAC (bacterial artificial chromosome). A BAC can replicate inside a bacterial cell. A set of BACs containing an entire human genome is called a BAC library.

The Clone-by-Clone StrategyMarkers used in mapping large genomesDifferent types of Markers are used in mapping large genomes, Such as A. Restriction Fragment Length Polymorphisms (RFLP) B. Variable Number of Tandem Repeats (VNTRs)C. Sequence Tagged Sites (STS)D. Microsatellites, etc.

A. Restriction Fragment Length Polymorphisms (RFLP) Polymorphism means that a genetic locus has different forms, or alleles. The cutting the DNA from any two individuals with a restriction enzyme may yield fragments of different lengths, called Restriction Fragment Length Polymorphisms (RFLP), is usually pronounced rifflip. The pattern of RFLP generated will depend mainly on1) The differentiation in DNA of selected strains (or) species2) The restriction enzymes used3) The DNA probe employed for southern hybridization

Steps: Consider the restriction enzyme HindIII, which recognizes the sequence AAGCTT. Between two, One individual contains three sites of a chromosome, so cutting the DNA with HindIII yields two fragments, 2 and 4 kb long.

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Figure: Detecting a RFLPAnother individual may lack the middle site but have the other two, so cutting the DNA with HindIII yields one fragment 6 kb long. These fragments are called RFLP. Continue

These restriction fragments of different lengths beteween the genotypes can be detected on southern blots and by the use of suitable probe. An RFLP is detected as a differential movement of a band on the gel lanes from different species and strains. Each such bond is regarded as single RFLP locus. So any differences among the DNA of individuals are easy to see.This RFLP is used as a marker in chromosomal mapping.

LimitationsRequires relatively large amount of highly pure DNALaborious and expensive to identify a suitable marker restriction enzymes.Time consuming.Required expertise in auto radiography because of using radio actively labeled probes

B. Variable Number of Tandem Repeats (VNTRs)Due to the greater the degree of polymorphism of a RFLP, mapping become very tedious, in this case variable number tandem repeats (VNTRs) will be more useful. Tandem repeatsoccur inDNAwhen a pattern of one or more nucleotides is repeated and the repetitions are directly adjacent to each other. An example would be:

In which the sequence ATTCGCCAATC is repeated three times.

Avariable number tandem repeat(orVNTR) is a location in agenomewhere a shortnucleotide sequenceis organized as atandem repeat.The repeated sequence is longer about 10-100 base pairs long.The full genetic profiles of individuals reveal many differences. Since most human genes are the same from person to person, but Variable Number of Tandem Repeats or VNTRs that tends to differ among different people.

ATTCGCCAATC ATTCGCCAATC ATTCGCCAATCContinue

While the repeated sequences themselves are usually the same from person to person, the number of times they are repeated tends to vary. VNTRs are highly polymorphic. These can be isolated from an individuals DNA and therefore relatively easy to map. However, VNTRs have a disadvantage as genetic markers: They tend to bunch together at the ends of chromosomes, leaving the interiors of the chromosomes relatively devoid of markers.

C. Sequence Tagged Sites (STS)

Another kind of genetic marker, which is very useful to genome mappers, is the sequence-tagged site (STS). STSs are short sequences, about 601000 bp long, that can be easily detected by PCR using specificprimers. The sequences of small areas of this DNA may be known or unknown, so one can design primers that will hybridize to these regions and allow PCR to produce double stranded fragments of predictable lengths. If the proper size appears, then the DNA has the STS of interest.One great advantage of STSs as a mapping tool is that no DNA must be cloned and examined. Instead, the sequences of the primers used to generate an STS are published and then anyone in the world can order those same primers and find the same STS in an experiment that takes just a few hours.

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In this example, two PCR primers (red) spaced 250 bp apart have been used. Several cycles of PCR generate many double-stranded PCR products that are precisely 250 bp long. Electrophoresis of this product allows one to measure its size exactly and confirm that it is the correct one.

Figure : Sequence-tagged sites

Geneticists interested in physically mapping or sequencing a given region of a genome aim to assemble a set of clones called a contig, which contains contiguous (actually overlapping) DNAs spanning long distances. It is essential to have vectors like BACs and YACs that hold big chunks of DNA. Assuming we have a BAC library of the human genome, we need some way to identify the clones that contain the region we want to map. A more reliable method is to look for STSs in the BACs. It is best to screen the BAC library for at least two STSs, spaced hundreds of kilo-bases apart, so BACs spanning a long distance are selected. After we have found a number of positive BACs, we begin mapping by screening them for several additional STSs, so we can line them up in an overlapping fashion as shown in following figure. This set of overlapping BACs is our new contig. We can now begin finer mapping, and even sequencing, of the contig.

Making physical map using Sequence Tagged Sites (STS) Continue

At top left, several representative BACs are shown, with different symbols representing different STSs placed at specific intervals. In step (a) of the mapping procedure, screen for two or more widely spaced STSs. In this case screen for STS1 and STS4. All those BACs with either STS1 or 4 are shown at top right. The identified STSs are shown in color. In step (b), each of these positive BACs is further screened for the presence of STS2, STS3, and STS5.The colored symbols on the BACs at bottom right denote the STSs detected in each BAC. In step (c), align the STSs in each BAC to form the contig. Measuring the lengths of the BACs by pulsed-field gel electrophoresis helps to pin down the spacing between pairs of BACs.

Fig: Mapping with STSs.

D. Microsatellites STSs are very useful in physical mapping or locating specific sequences in the genome. But sometimes it is not possible to use them for genetic mapping. Fortunately, geneticists have discovered a class of STSs called microsatellites.

GCTTGGTGTGATGTAGAAGGCGCCAATGCATCTCGACGTATGCGTATACGGGTTACCCCCTTTGCAATCAGTGCACACACACACACACACACACACACACACACACACACACAGTGCCAAGCAAAAATAACGCCAAGCAGAACGAAGACGTTCTCGAGAACACC

Microsatellites are similar to minisatellites in that they consist of a core sequence repeated over and over many times in a row. The core sequence in typical microsatellites is smallerusually only 24 bp long. Microsatellites are highly polymorphic; they are also widespread and relatively uniformly distributed in the human genome.The number of repeats varied quite a bit from one individual to another.Thus, they are ideal as markers for both linkage and physical mapping.

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In 1992, Jean Weissenbach et al produced a linkage map of the entire human genome based on 814 microsatellites containing a CA dinucleotide repeat.

The most common way to detect microsatellites is to design PCR primers that are unique to one locus in the genome and unique on base pair on either side of the repeated portion. Therefore, a single pair of PCR primers will work for every individual in the species and produce different sized products for each of the different length microsatellites.The PCR products are then separated by either gel electrophoresis. Either way, the investigator can determine the size of the PCR product and thus how many times the dinucleotide ("CA") was repeated for each allele.

Whole genome Shotgun SequencingThe shotgun-sequencing strategy, first proposed by Craig Venter, Hamilton Smith, and Leroy Hood in 1996, bypasses the mapping stage and goes right to the sequencing stage. This method was employed by Celera Genomics, which was a private entity that was trying to mono-polise the human genome sequence by patenting it, to do this they had to try and beat the publicly funded project. Whole genome shotgun sequencing was therefore adopted by them.

1. BAC library: A BAC library is generated of random fragments of the human genome using restriction digestion followed by cloning.The sequencing starts with a set of BAC clones containing very large DNA inserts, averaging about 150 kb. The insert in each BAC is sequenced on both ends using an automated sequencer that can usually read about 500 bases at a time, so 500 bases at each end of the clone will be determined. Assuming that 300,000 clones of human DNA are sequenced this way, that would generate 300 million bases of sequence, or about 10% of the total human genome. These 500-base sequences serve as an identity tag, called a sequence-tagged connector (STC), for each BAC clone. This is the origin of the term connectoreach clone should be connected via its STCs to about 30 other clones.Continue

Fig: Whole Genome Shotgun Sequencing MethodSteps:1. BAC library2. Finger printing3. Plasmid library4. BAC walking5. Powerful computer programContinue

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2. Finger printing: This step is to fingerprint each clone by digesting it with a restriction enzyme. This serves two important purposes. First, it tells the insert size (the sum of the sizes of all the fragmented by the restriction enzyme). Second, it allows one to eliminate aberrant clones whose fragmentation patterns do not fit the consensus of the overlapping clones. Note that this clone fingerprinting is not the same as mapping; it is just a simple check before sequencing begins.3. Plasmid library: A seed BAC is selected for sequencing. The seed BAC is sub cloned into a plasmid vector by subdividing the BAC into smaller clones only about 2 kb. A plasmid library is prepared by transforming E. coli strains with plasmid. This whole BAC sequence allows the identification of the 30 or so other BACs that overlap with the seed: They are the ones with STCs that occur somewhere in the seed BAC.

4. BAC walking: Three thousand of the plasmid clones are sequenced, and the sequences are ordered by their overlaps, producing the sequence of the whole 150-kb BAC. Finding the BACs (about 30) with overlapping STCs, then compare them by fingerprinting to find those with minimal overlaps, and sequence them. This strategy, called BAC walking, would in principle allow one laboratory to sequence the whole human genome.

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5. Powerful computer program: But we do not have that much time, so Venter and colleagues modified the procedure by sequencing BACs at random until they had about 35 billion bp of sequence. In principle that should cover the human genome ten times over, giving a high degree of coverage and accuracy. Then they fed all the sequence into a computer with a powerful program that found areas of overlap between clones and fit their sequences together, building the sequence of the whole genome.

Finishing Process of assembling raw sequence reads into accurate contiguous sequenceRequired to achieve 1/10,000 accuracyManual processLook at sequence reads at positions where programs cant tell which base is the correct oneFill gapsEnsure adequate coverage

GapSinglestrandedContinue

Although automated editing programs like PHRAP have greatly increased the efficiency of sequencing, there remains a need for human judgment and intervention. This occurs during the finishing step, which is defined as the process of assembling the raw sequence reads into an accurate contiguous genomic sequence. For genomic sequencing with an accuracy of one error in 10,000 bases, a manual finishing step is essential. The finisher looks at positions where the automated editing program cant tell which base is the correct one. By examining the various raw sequence reads, the finisher then makes a judgment call as to the correct base or sends the region back for additional sequencing. Similarly, when there are gaps in the sequence or insufficient coverage, the finisher will flag the region and send it back to the production sequencing team for more work.

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Finishing To fill gaps in sequence, design primers and sequence from primerTo ensure adequate coverage, find regions where there is not sufficient coverage and use specific primers for those areas

GAP

PrimerPrimer

Gaps are usually filled by designing custom sequencing primers that are complementary to the regions adjacent to the gap. The sequencing reaction is then performed using these custom primers on a clone containing the problematic region of DNA as the template. A similar strategy is used for regions with insufficient coverage: Custom primers are made and then used for directed sequencing of a particular region.

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VerificationRegion verified for the following:CoverageSequence qualityContiguityDetermine restriction-enzyme cleavage sites Generate restriction map of sequenced regionMust agree with fingerprint generated of clone during mapping step

The final step of finishing is to verify the sequence. All regions are checked for the extent of coverage (i.e., how many times the same region has been sequenced, and in what direction), for sequence quality (i.e., whether ambiguity has been removed for all positions in the sequence), and for contiguity (i.e., whether the sequence forms one uninterrupted stretch of DNA). A good test of sequence quality that is frequently used in the finishing stage is to determine the sites where restriction enzymes would cut in the newly acquired sequence. A restriction map is generated from the sequence and then compared with the known fingerprint generated from the clone during the mapping step. If both show the same pattern, then it is considered to be an indication that the sequence is of high quality and relatively error free.

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HISTORY OF DNA SEQUENCING1972 Earliest nucleotide sequencing RNA sequencing of Bacteriophage MS2 by WALTER FIESERREarly sequencing was performed with tRNA through a technique developed by Richard Holley, who published the first structure of a tRNA in 1964.

1977 - DNA sequencing FREDRICK SANGER by Chain termination method

Chemical degradation method by ALLAN MAXAM and WALTER GILBERT

1977 - First DNA genome t be sequenced of Bacteriophage X174

1986 - LOREY and SMITH gave Semiautomated sequencing 1987 Applied biosystems marketed Fully automated sequencing machines

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Determining the Sequence of DNAMethods:

Maxam and Gilbert chemical degradation methodChain termination or Dideoxy methodFredrick SangerGenome sequencing method Shotgun sequencing Clone contig approach2nd generation sequencing methodsPyrosequencingNanopore sequencingIllumina sequencingSolid sequencing

SANGER SEQEUNCINGChain termination method of DNA sequencing.It involves following components: 1. Primer 2. DNA template 3. DNA polymerase 4.. dNTPs(A,T,G,C) 5. ddNTPs

4 Steps:Denaturation Primer attachment and extension of basesTerminationPoly acrylamide gel electrophoresis

SANGERS METHOD

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ddATP + ddAfour dNTPs dAdGdCdTdGdCdCdCdG

ddCTP + dAdGddCfour dNTPs dAdGdCdTdGddC dAdGdCdTdGdCddC dAdGdCdTdGdCdCddC

ddGTP + dAddGfour dNTPs dAdGdCdTddG dAdGdCdTdGdCdCdCddG

ddTTP + dAdGdCddTfour dNTPs dAdGdCdTdGdCdCdCdGA

C

G

TChain Termination (Sanger) Sequencing

Determination of nucleotide sequence

SANGERS METHODNot all polymerases can be used as they have mixed activity of polymerizing and degrading.Both exonuclease activities are detrimental.Klenow fragment was used in orignal method but it has low processivity.So Sequenase from bacteriophage T7 was uesd with high processivity and no exonuclease added.Method requires ss DNA. So it is obtained byDenaturation with alkali or boilingDNA can be cloned in phagemid containg M13 ori and can take up DNA fragments of 10kb

PYROSEQUENCINGPyrosequencing is the second important type of DNA sequencing methodology in use today.

The addition of a DNTP is accompanied by release of a molecule of pyrophosphate.

Reaction mixture contains DNA sample to be sequenced Primers Deoxynucleotides DNA polymerase Sulfurylase

The release of pyrophosphate is converted by the enzyme sulfurylase into a flash of chemiluminescence which is easily automated.

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Animated videos of Sangers and Pyrosequencing methods for better understanding:

PYROSEQUENCINGAdvantages:AccurateParallel processingEasily automatedEliminates the need for labeled primers and nucleotidesNo need for gel electrophoresis DISADVANTAGESSmaller sequencesNonlinear light response after more than 5-6 identical nucleotides

MASSIVELY PARALLEL PYROSEQUENCINGThe DNA is broken down into fragments between 300 to 500bpEach fragment is ligated with a pair of adaptorTo attach to the beads Provide annealing sites for the primers for performing PCRAdaptors are attached to beads by biotin-streptavidin linkageJust one fragment becomes attached to one beadEach DNA fragment is now amplified usingPCR is carried out in a oil emulsion, each bead residing within own droplet in the emulsionEach droplet contains all the reagents for PCR and is physically seprated from all the other droplets by the barrier provided by the oil components in the emulsion.After PCR, the droplets are transferred on wells on plastic strip and pyrosequencing reactions are carried out

SHOTGUN SEQUENCING Shotgun sequencing, also known asshotgun cloning, is a method used forsequencinglongDNAstrands or the whole genome.

In shotgun sequencing,DNA is broken up randomly into numerous small segments and overlapping regions are identified between all the individual sequences that are generated.

Multiple overlapping reads for the target DNA are obtained by performing several rounds of this fragmentation and sequencing.

Computer programs then use the overlapping ends of different reads to assemble them into a continuous sequence.

The shotgun approach was first used successfully with the bacterium Haemophilus influenzae.

Craig venter used this method to map the Human genome project in 2001.

Shotgun sequencing

Shotgun sequencing AND Next Generation Sequencing ANIMATED VIDEOS for better understanding:

NEXT GENERATION SEQUENCINGThe concept behind NGS the bases of small fragments of DNAare sequentially identifed as signals emitted as eachfragment is resynthesized from a dna template strand NGS extends this process across millions of reactions in a massively parallel fashion rather than being limited to a single or a few dna fragments

Illumina sequencing

Illumina sequencing

SOLiD SEQUENCING

Illumina Sequencing Solid Sequencing animated VIDEOS:

SOLiD SEQUENCINGThe SOLiD instrument utilizes a series of ligation and detection rounds to sequence millions of fragments simultaneously. There are five primer cycles performed on the instrument with each cycle staggered by a single base and including a series of seven or ten ligations for either a 35 or 50 base pair sequencing run. Each ligation decodes two bases and is recorded through fluorescent imaging. By compiling the fluorescent reads in color space for each fragment, an accurate sequence can be generated.Two types of libraries are available for sequencing Fragment and Mate Pairs.

HUMAN GENOME PROJECT SHORT VIDEOS OVERVIEW:

Conclusion:The project was not able to sequence all the DNA found in human cells. It sequenced only "euchromatic" regions of the genome, which make up more than 95% of the genome. The other regions, called "heterochromatic" are found in centromeres and telomeres, and were not sequenced under the project.

The Human Genome Project was declared complete in April 2003. An initial rough draft of the human genome was available in June 2000 and by February 2001 a working draft had been completed and published followed by the final sequencing mapping of the human genome on April 14, 2003. Although this was reported to cover 99% of the euchromatic human genome with 99.99% accuracy, a major quality assessment of the human genome sequence was published on May 27, 2004 indicating over 92% of sampling exceeded 99.99% accuracy which was within the intended goal. Further analyses and papers on the HGP continue to occur.

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