1000G and UK10K publish results of large scale human genome sequencing

Both 1000G and UK10K consortia have recently published the results of their analysis on the variability of human genomes, based on their large scale genomics projects.

In the 1000G papers appeared on Nature the consortium described SNV and structural variants findings based on the phase 3 dataset. 

Citing the abstract, they have analyzed "2,504 individuals from 26 populations using a combination of low-coverage whole-genome sequencing, deep exome sequencing, and dense microarray genotyping. We characterized a broad spectrum of genetic variation, in total over 88 million variants (84.7 million single nucleotide polymorphisms (SNPs), 3.6 million short insertions/deletions (indels), and 60,000 structural variants), all phased onto high-quality haplotypes. This resource includes >99% of SNP variants with a frequency of >1% for a variety of ancestries."

You can find the papers here:

A global reference for human genetic variation (Nature, 2015)

An integrated map of structural variation in 2,504 human genomes (Nature, 2015)

The UK10K consortium also published a detailed description of the human genetic variability based on around 10,000 samples, partially low coverage WGS of control samples and partially deep covered WES focused on various complex and rare diseases.

Citing the abstract, "Here we describe insights from sequencing whole genomes (low read depth, 7×) or exomes (high read depth, 80×) of nearly 10,000 individuals from population-based and disease collections. In extensively phenotyped cohorts we characterize over 24 million novel sequence variants, generate a highly accurate imputation reference panel and identify novel alleles associated with levels of triglycerides (APOB), adiponectin (ADIPOQ) and low-density lipoprotein cholesterol (LDLR and RGAG1) from single-marker and rare variant aggregation tests. We describe population structure and functional annotation of rare and low-frequency variants, use the data to estimate the benefits of sequencing for association studies, and summarize lessons from disease-specific collections."

By the way, they published also an improved haplotype reference panel that can be used to improve imputation of low-frequency and rare variants also developed an online tools to explore their association results.

The third paper is a first example of the disease-oriented results obtained by the consortium: they identified EN1 as a gene involved in reduced bone density and recurrent fracture.

You can find the papers here:

The UK10K project identifies rare variants in health and disease (Nature, 2015)

Improved imputation of low-frequency and rare variants using the UK10K haplotype reference panel (Nature Communications, 2015)

Whole‐genome sequencing identifies EN1 as a determinant of bone density and fracture (Nature, 2015)

Exome Aggregation Consortium release its data on 63,000 exomes!

On October 29th, the Exome Aggregation Consoritum as released its browser based on the impressive number of 63,000 human exomes.
This database is the larger collection of human exome data so far and provide both a web base interface to retrieve variants in your gene of interest or the download of a VCF file containing the list of all the annotated variants.

The final dataset is based on sequences from several consortia working on complex disorders and also includes 1000G and ESP6500 data.

The first aim of the Consortium is to study the distribution of "human knockout", that is people having both copies of a given gene inactivated by severe mutations. The analysis of associated phenotype data promise to reveal lot of interesting information of the actual role of single human genes. Moreover, the study of subjects carrying inactivating mutations on known disease genes but not showing the expected phenotype could lead to identification of new therapeutic targets.

See more information on Nature News and Genome Web!

First user reports on Oxford Nanopore MinION!

After the start of the early access program, the sequencing community is waiting for the first results and comments on the MinION platform by Oxford Nanopore. This sequencer promises to revolutionize the field and is the first nanopore based sequencer that have reached the market.

Nick Loman, one of the early customer has now reported the first results obtained on the new platform. It is a 8.5 Kb read from P. aeruginosa showing that MinION can produce useful data, even if the accuracy remains low. Analyses of a read by two bioinformatics researchers, who used different alignment tools and posted their results here and here, showed that the read is about 68 percent identical to the P. aeruginosa genome and has many errors, particularly gaps. Main issues seems to be in the basecalling software, but Oxford Nanopore is working hard to improve it. Accordingly also to Konrad Paszkiewicz, another early customer, the device itself is really robust and easy to use and the library preparation procedure is simple, resulting in low sequencing costs.

The mean read length seems to be about 10 Kb, but users reported even longer reads up to 40 Kb, covering the entire lambda genome used for testing. So the read length is really promising and place the mature MinION as a natural competitor to PacBio.

The use of MinION seems straightforward: after plugging the sequencer into a USB 3.0 port of a computer, it installs the MinKnow software suite. A program called Metrichor uploads the raw data – ion current traces – to the Amazon Elastic Compute Cloud, where base-calling happens, either 1D base-calling for unidirectional reads or 2D base-calling for bidirectional reads.
Overall, improvements have to be made to the base-calling software, reliability of the flow cells, and library shelf-life, and new software needs to be developed by the community to take advantage of the MinION reads.  Oxford Nanopore said a new chemistry will be available in the coming months, which might include some of these improvements.

In the meantime, many other early access users contacted by IS website are awaiting the arrival of reagents, are in the midst of burn-in, or have run their own samples but are not ready to talk about their results yet. So we are expecting many more data and comments and detailed estimation on platform accuracy and data production to be out in the next months! The new minion has fulfilled the excpection in this first test and there is a lot promising about this new technology...maybe a new revolution in the field is ready to come!

Other details can be found on this post on GenomeWeb.

Illumina presents two new sequencing platforms: population scale genomics and the 1000$ genome

Few days ago Illumina announced two new NGS platforms: a huge factory scale sequencer called HiSeq X Ten and a new benchtop sequencher called NextSeq 500, half-way between a MiSeq and a HiSeq.

Both platforms represent a huge advancement in data production, made possible by several technical innovations and a new chemistry. First of all, Illumina worked hard to increase accuracy and speed of image acquisition using an increased number (up to 6) of new LED cameras for image snapshot and a new flow cell design with larger random clusters enabling it to work with the lower-resolution optics as well as new surface chemistry to enhance the signal.

The HiSeq X Ten will also integrate a dual direction image scan system dubbling the scan speed and a new flow cell containing nanowell that allow for a precise cluster separation resulting in more dense clustering.

Both instruments will run with the new 2-color chemistry. This methods use only 2 different fluorescent molecule, red and green: T and C bases are marked as green or red signal, respectively; A is marked with both signal and G lack any marker. Thus, only 2 image acquisitions, one per color channel, are needed every cycle, instead of the classic 4, cutting down the processing time. The chemistry is well explained in CoreGenomics blog and the Illumina tech sheet.

  

The NextSeq 500 will come together with two diffent flowcell and two different run mode, resembling the fast run mode of the HiSeq 2500.

The mid-output flow cell includes 130 million clusters and will support a 2x75 base kit that will generate 16-19 gigabases of data per 15-hour run, or a 2x150 base kit that will generate 32-39 gigabases of data in a 26-hour run.
The high-output flow cell includes 400 million clusters and will support a 2x150 base kit that will generate 100-120 gigabases of data per 29-hour run, a 2x75 base kit that will generate 50-60 gigabases per 18-hour run, and a 1x75 base kit that will generate 25-30 gigabases per 11-hour run.

This numbers will allow a WGS sequencing in little more than a day at about 30X, making the NextSeq 500 the first benchtop sequencer to hit the goal of whole genome.

The HiSeq X Ten is a huge sequencher and it's actually composed by 10 single sequencing unit that will cost you a total of 10M$. Illumina will accept only a minimum order of ten units, with each supplementary unit costing 1M$. One unit will be able to generate 600 gigabases of data in one day, enough to sequence five human genomes, or 1.8 terabases of data in under three days, so that the total data production will be 18Tb every 3 days, allowing the sequencing of 18000 genome every year!!

Illumina claims that this juggernaut will respond to the need of population scale sequencing programs, often national health programs, such as UK initiative to sequence 100000 individuals or the Denmark project to sequence the entire population of an isolated island.

The HiSeq X Ten will enable the "first real $1,000 genome," said Flatley, CEO of Illumina. One reagent kit to support 16 genomes per run will cost $12,700, or $800 per genome for reagents. Hardware will add an additional $137 per genome, while sample prep will range between $55 and $65 per genome.

However, the new machine will sequence ONLY whole human genomes, no other applications are supported by now, and, given the hard work needed to produce and set up such a huge instruments, Illumina will deliver only 5 of them in the first year.

Despite the 10M$ price, Illumina has already sold 4 HiSeq X Ten: to the South Korean sequencing service provider Macrogen, the Garvan Institute in Australia, the New York Genome Center, and to the Broad Institute, which purchased a 14-unit system.

Detailed information and interesting discussions around the two new platforms and their technical innovations can be found around the web: CoreGenomics (presentation, HiSeq X Ten, NextSeq 500), MassGenomics, Omics!Omics!, Opniomics, GenomeWeb, Nature News. 

So is the mythical goal of 1000$ genome finally achieved? Well, it seems almost...

First of all, one have to consider the initial investment and the overhead costs to run the 10 machines. Moreover, the cost estimate made by Illumina are based on 4 year full activity of the HiSeq X Ten, which means 18.000 genomes per year per 4 year with machine running 24h/day...This scenario seems unlikely to many experts, since we simply don't have so many samples to sequence.

Finally data analysis costs, besides the simple sequence alignment and maybe SNP call, are not included as usually. For a more detailed evaluation of the real costs, read the interesting post on allseq blog.

Happy new Human Genome! GRCh38 is here!

This Christmas bring a long desired gift for anyone involved in genomics: a brand new release of the Human Genome Assembly. The new GRCh38 is a kind gift from the Genome Research Consortium (GRC), including The Wellcome Trust Sanger Institute (WTSI), the Washington University Genome Sciences Center (WUGSC), the European Bioinformatics Institute (EBI) and the The National Center for BiotechnologyInformation (NCBI). This new version is the first major release since 4 years and provide two major improvements: fewer gaps and centromere model sequences.

 




The first draft of the human genome contained around 150000 gaps, years of hard work have reduced them to 357 in the GRCh37 version. The new GRCh38 take care of several of these gaps, including one on chromosome 10 associated with the mannose receptor C Type 1 (MRC1) locus and one on chromosome 17, associated with the chemokine (C-C-motif) ligand 3 like 1 and ligand 4 like 1 (CCL3L1/CCL4L1 ) genes. In addition, there have been additions from whole-genome shotgun sequencing at nearly 100 of GRCh37′s assembly gaps.
Telomeres continue to be represented by default 10 kilobase gaps, while some improvments have been done on acrocentric chromosomes and GRCh38 includes new sequences on the short arms of chromosomes 21 and 22.

The other major feature added to the new release is the model sequence representation for centromeres and some heterochromatin. Using a method developed by a research team at University of California at Santa Cruz (UCSC) and reads generated during the Venter genome assembly, scientists created models for the centromeres. “These models don’t exactly represent the centromere sequences in the Venter assembly, but they are a good approximation of the ‘average’ centromere in this genome” says Church, a genomicist formerly at the US National Center for Biotechnology Information. Even if these sequence models are not exact representations of any real centromere, they will likely improve genome analysis and allow study of variation in centromere sequences.

GRC recently submitted the data for GRCh38 to GenBank, and the assembly is available with accession GCA_000001405.15. These data are also available by FTP at ftp.ncbi.nlm.nih.gov/genbank/genomes/Eukaryotes/vertebrates_mammals/Homo_sapiens/GRCh38.

However keep in mind that this sequence is provided withou any annotation and that it will take at least a couple of week for the NCBI annotation pipeline to process the whole data and produce a new set of RefSeqs. As the NCBI reports: "The chromosome sequences will continue to have accessions NC_000001-NC_000024, but their versions will update as GRCh38 includes a sequence change for all chromosomes. This process generally takes about 2 weeks, and when that is done we will incorporate these sequences into various analysis and display tools, such as genomic BLAST and genome viewers. Thus, at the end of this process each chromosome will be represented by both an unannotated sequence in GenBank (the original GRC data) and an annotated sequence in the RefSeq collection."

Further details on the properties and development of the GRCh38 are reported by Methagora blog from Nature Methods, NCBI Insights blog and the GRC consotium blog.

Flash Report: Oxford Nanopore is announcing an early access program for its MinIon nanopore sequencer

The MinION is a USB stick (!!!) DNA sequencer announced by Oxford Nanopore Technologies about one year and a half ago. The company, which is currently exhibiting at the American Society of Human Genetics, yesterday announced the launch of the MinION Access Program.
More details in this GenomeWeb article as well as on this post on Nick Loman's blog. Interesting insights into the DNA sample preparation have been posted on the Future Continuous blog.

MinION Access Programme
In late November, Oxford Nanopore will open registration for a MinION Access Programme (MAP – product preview). This is a substantial but initially controlled programme designed to give life science researchers access to nanopore sequencing technology at no risk and minimal cost.
MAP participants will be at the forefront of applying a completely novel, long-read, real-time sequencing system to existing and new application areas. MAP participants will gain hands-on understanding of the MinION technology, its capabilities and features. They will also play an active role in assessing and developing the system over time. Oxford Nanopore believes that any life science researcher can and should be able to exploit MinION in their own work. Accordingly, Oxford Nanopore is accepting applications for MAP participation from all1, 2.
About the programme
A substantial number of selected participants will receive a MinION Access programme package. This will include:
* At least one complete MinION system (device, flowcells and software tools).
* MAP participants will be asked to pay a refundable $1,000 deposit on the MinION USB device, plus shipping.
* Oxford Nanopore will provide a regular baseline supply of flowcells sufficient to allow frequent usage of the system. MAP participants will ONLY pay shipping costs on these flowcells. Any additional flowcells required at the participants’ discretion may be available for purchase at a MAP-only price of $999 each plus shipping and taxes.
* Oxford Nanopore will provide Sequencing Preparation Kits. MAP participants may choose to develop their own sample preparation and analysis methods; however, at this stage on an unsupported basis.
What are the terms of the MAP agreement?
Participation in the MAP product preview program will require participants to sign up to an End User License Agreement (EULA) and simple terms intended to allow Oxford Nanopore to further develop the utility of the products, applications and customer support while also maximising scientific benefits for MAP participants. Further details will be provided when registration opens, however in outline:
* MAP participants will be invited to provide Oxford Nanopore with feedback regarding their experiences through channels provided by the company.
* All used flow cells are to be returned to Oxford Nanopore3.
* MAP participants will receive training and support through an online participant community and support portal.
* MAP participants will go through an initial restricted ‘burn-in’ period, during which test samples will be run and data shared with Oxford Nanopore. After consistent and satisfactory performance has been achieved under pre-agreed criteria, the MAP participants will be able to conduct experiments with their own samples. Data can be published whilst participants are utilising the baseline supply of flowcells.
* MAP participants or Oxford Nanopore may terminate participation in the programme at any time, for any reason. Deposits will be refunded after all of the MAP hardware is returned.
* MAP participants will be the first to publish data from their own samples. Oxford Nanopore does not intend to restrict use or dissemination of the biological results obtained by participants using MinIONs to analyse their own samples. Oxford Nanopore is interested in the quality and performance of the MiniION system itself.
* Oxford Nanopore intends to give preferential status for the GridION Access Programme (GAP) when announced to successful participants in the MinION access programme.
* The MinION software will generate reports on the quality of each experiment and will be provided to Oxford Nanopore only to facilitate support and debugging.
Registration process
Registration will open in late November for a specific and limited time period. Oxford Nanopore will operate a controlled release of spaces on the programme.
MAP participants will be notified upon acceptance to the programme. They will then able to review and accept the EULA before providing the refundable deposit and joining the programme. MAP participants will then receive a login for the participant support portal and a target delivery date for their MinION(s) and initial flow cells.
The online participant support portal will provide training materials, FAQs, support and other information such as data examples from Oxford Nanopore. It will also include a community forum to allow participants to share experiences.
Who can join?
Anybody who is not affiliated with competitors of Oxford Nanopore. Strong preference will be given to biologists/researchers working within the field of applied NGS where long reads, simple workflow, low costs, and real time analysis can be shown to make a key difference. Preference may also be given to individuals/sites opting for multiple MinIONs. If the programme is oversubscribed, some element of fairly applied random selection may be used to further prioritise participants.
1. If you would like us to keep you informed of the opening of this registration please visit our contact page and select the box marked ‘Keep me informed on the MinION Access programme’.
2. The MinION system is for Research Use Only
3. Flowcells can be easily, quickly and thoroughly washed through with water and dried before return.

Breaking News: Bigfoot genome finally published

It's not April 1st yet, however something a little bit weird has been reported on several web sites.
Let's start from the beginning: back in November 2012 a team of researchers (experts in genetics, forensics, imaging, and pathology led by Dr. Melba S. Ketchum) claimed that through a DNA study they were able to sequence three complete Bigfoot (also known as Sasquatch) nuclear genomes and determined that the species is a human hybrid. The scientists said that Bigfoot is a human relative that arose roughly 15,000 years ago and is a hybrid of modern Homo sapiens and an unknown primate species.
A few days ago a paper describing the genome sequence was published on an online journal called De Novo (apparently the team performing the study had some trouble publishing their results so they decided to... buy an existing journal and renamed it). Unfortunately the scientists are trying to charge $30 to anyone wanting to view their research paper. Fortunately somebody was able to get their hands on a copy of the paper and found out that...

You can read more about it in this Ars Technica article.

Below is the abstract of the paper that, strange enough, cannot be found in PubMed.

Ketchum, M. S., Wojtkiewicz, P. W., Watts, A. B., Spence, D. W., Holzenburg, A. K., Toler, D. G., Prychitko, T. M., Zhang, F., Bollinger, S., Shoulders, R., Smith, R. 

Novel North American Hominins, Next Generation Sequencing of Three Whole Genomes and Associated Studies

DeNovo Scientific Journal, 2013, Vol. No. 1

One hundred eleven samples of blood, tissue, hair, and other types of specimens were studied, characterized and hypothesized to be obtained from elusive hominins in North America commonly referred to as Sasquatch. DNA was extracted and purified from a subset of these samples that survived rigorous screening for wildlife species identification. Mitochondrial DNA (mtDNA) sequencing, specific genetic loci sequencing, forensic short tandem repeat (STR) testing, whole genome single nucleotide polymorphism (SNP) bead array analysis, and next generation whole genome sequencing were conducted on purported Sasquatch DNA samples gathered from various locations in North America. Additionally, histopathologic and electron microscopic examination were performed on a large tissue sample. vel non-human DNA.