Functional equivalence of genome sequencing analysis pipelines enables harmonized variant calling across human genetics projects.

Hundreds of thousands of human whole genome sequencing (WGS) datasets will be generated over the next few years. These data are more valuable in aggregate: joint analysis of genomes from many sources increases sample size and statistical power. A central challenge for joint analysis is that different WGS data processing pipelines cause substantial differences in variant calling in combined datasets, necessitating computationally expensive reprocessing. This approach is no longer tenable given the scale of current studies and data volumes. Here, we define WGS data processing standards that allow different groups to produce functionally equivalent (FE) results, yet still innovate on data processing pipelines. We present initial FE pipelines developed at five genome centers and show that they yield similar variant calling results and produce significantly less variability than sequencing replicates. This work alleviates a key technical bottleneck for genome aggregation and helps lay the foundation for community-wide human genetics studies.

Empowering genomic medicine by establishing critical sequencing result data flows: the eMERGE example.

The eMERGE Network is establishing methods for electronic transmittal of patient genetic test results from laboratories to healthcare providers across organizational boundaries. We surveyed the capabilities and needs of different network participants, established a common transfer format, and implemented transfer mechanisms based on this format. The interfaces we created are examples of the connectivity that must be instantiated before electronic genetic and genomic clinical decision support can be effectively built at the point of care. This work serves as a case example for both standards bodies and other organizations working to build the infrastructure required to provide better electronic clinical decision support for clinicians.

Spontaneous shaker rat mutant - a new model for X-linked tremor/ataxia.

The shaker rat is an X-linked recessive spontaneous model of progressive Purkinje cell (PC) degeneration exhibiting a shaking ataxia and wide stance. Generation of Wistar Furth (WF)/Brown Norwegian (BN) F1 hybrids and genetic mapping of F2 sib-sib offspring using polymorphic markers narrowed the candidate gene region to 26 Mbp denoted by the last recombinant genetic marker DXRat21 at 133 Mbp to qter (the end of the long arm). In the WF background, the shaker mutation has complete penetrance, results in a stereotypic phenotype and there is a narrow window for age of disease onset; by contrast, the F2 hybrid phenotype was more varied, with a later age of onset and likely non-penetrance of the mutation. By deep RNA-sequencing, five variants were found in the candidate region; four were novel without known annotation. One of the variants caused an arginine (R) to cysteine (C) change at codon 35 of the ATPase, Ca(2+) transporting, plasma membrane 3 (Atp2b3) gene encoding PMCA3 that has high expression in the cerebellum. The variant was well supported by hundreds of overlapping reads, and was found in 100% of all affected replicas and 0% of the wild-type (WT) replicas. The mutation segregated with disease in all affected animals and the amino acid change was found in an evolutionarily conserved region of PMCA3. Despite strong genetic evidence for pathogenicity, in vitro analyses of PMCA3(R35C) function did not show any differences to WT PMCA3. Because Atp2b3 mutation leads to congenital ataxia in humans, the identified Atp2b3 missense change in the shaker rat presents a good candidate for the shaker rat phenotype based on genetic criteria, but cannot yet be considered a definite pathogenic variant owing to lack of functional changes.

External quality assessment: best practice.

There is a requirement for accredited laboratories to participate in external quality assessment (EQA) schemes, but there is wide variation in understanding as to what is required by the laboratories and scheme providers in fulfilling this. This is not helped by a diversity of language used in connection with EQA; Proficiency testing (PT), EQA schemes, and EQA programmes, each of which have different meanings and offerings in the context of improving laboratory quality. We examine these differences, and identify what factors are important in supporting quality within a clinical laboratory and what should influence the choice of EQA programme. Equally as important is how EQA samples are handled within the laboratory, and how the information provided by the EQA programme is used. EQA programmes are a key element of a laboratory's quality assurance framework, but laboratories should have an understanding of what their EQA programmes are capable of demonstrating, how they should be used within the laboratory, and how they support quality. EQA providers should be clear as to what type of programme they provide - PT, EQA Scheme or EQA Programme.

Comparative analyses of human single- and multilocus tandem repeats.

Using the compiled human genome sequence, we systematically cataloged all tandem repeats with periods between 20 and 2000 bp and defined two subsets whose consensus sequences were found at either single-locus tandem repeats (slTRs) or multilocus tandem repeats (mlTRs). Parameters compiled for these subsets provide insights into mechanisms underlying the creation and evolution of tandem repeats. Both subsets of tandem repeats are nonrandomly distributed in the genome, being found at higher frequency at many but not all chromosome ends and internal clusters of mlTRs were also observed. Despite the integral role of recombination in the biology of tandem repeats, recombination hotspots colocalized only with shorter microsatellites and not the longer repeats examined here. An increased frequency of slTRs was observed near imprinted genes, consistent with a functional role, while both slTRs and mlTRs were found more frequently near genes implicated in triplet expansion diseases, suggesting a general instability of these regions. Using our collated parameters, we identified 2230 slTRs as candidates for highly informative molecular markers.