fastp an ultra-fast all-in-one FASTQ preprocessor, bioRxiv, 2018-03-02

AbstractMotivationQuality control and preprocessing of FASTQ files are essential to providing clean data for downstream analysis. Traditionally, a different tool is used for each operation, such as quality control, adapter trimming, and quality filtering. These tools are often insufficiently fast as most are developed using high-level programming languages (e.g., Python and Java) and provide limited multi-threading support. Reading and loading data multiple times also renders preprocessing slow and IO inefficient.ResultsWe developed fastp as an ultra-fast FASTQ preprocessor with useful quality control and data-filtering features. It can perform quality control, adapter trimming, quality filtering, per-read quality cutting, and many other operations with a single scan of the FASTQ data. It also supports unique molecular identifier preprocessing, poly tail trimming, output splitting, and base correction for paired-end data. It can automatically detect adapters for single-end and paired-end FASTQ data. This tool is developed in C++ and has multi-threading support. Based on our evaluation, fastp is 2–5 times faster than other FASTQ preprocessing tools such as Trimmomatic or Cutadapt despite performing far more operations than similar tools.Availability and ImplementationThe open-source code and corresponding instructions are available at <jatsext-link xmlnsxlink=httpwww.w3.org1999xlink ext-link-type=uri xlinkhref=httpsgithub.comOpenGenefastp>httpsgithub.comOpenGenefastp<jatsext-link>Contactchen@haplox.com

biorxiv bioinformatics 100-200-users 2018

In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations, bioRxiv, 2018-02-28

CRISPR-Cas genome-editing nucleases hold substantial promise for human therapeutics1–5 but identifying unwanted off-target mutations remains an important requirement for clinical translation6, 7. For ex vivo therapeutic applications, previously published cell-based genome-wide methods provide potentially useful strategies to identify and quantify these off-target mutation sites8–12. However, a well-validated method that can reliably identify off-targets in vivo has not been described to date, leaving the question of whether and how frequently these types of mutations occur. Here we describe Verification of In Vivo Off-targets (VIVO), a highly sensitive, unbiased, and generalizable strategy that we show can robustly identify genome-wide CRISPR-Cas nuclease off-target effects in vivo. To our knowledge, these studies provide the first demonstration that CRISPR-Cas nucleases can induce substantial off-target mutations in vivo, a result we obtained using a deliberately promiscuous guide RNA (gRNA). More importantly, we used VIVO to show that appropriately designed gRNAs can direct efficient in vivo editing without inducing detectable off-target mutations. Our findings provide strong support for and should encourage further development of in vivo genome editing therapeutic strategies.

biorxiv molecular-biology 100-200-users 2018

Complete assembly of parental haplotypes with trio binning, bioRxiv, 2018-02-27

AbstractReference genome projects have historically selected inbred individuals to minimize heterozygosity and simplify assembly. We challenge this dogma and present a new approach designed specifically for heterozygous genomes. “Trio binning” uses short reads from two parental genomes to partition long reads from an offspring into haplotype-specific sets prior to assembly. Each haplotype is then assembled independently, resulting in a complete diploid reconstruction. On a benchmark human trio, this method achieved high accuracy and recovered complex structural variants missed by alternative approaches. To demonstrate its effectiveness on a heterozygous genome, we sequenced an F1 cross between cattle subspecies Bos taurus taurus and Bos taurus indicus, and completely assembled both parental haplotypes with NG50 haplotig sizes &gt;20 Mbp and 99.998% accuracy, surpassing the quality of current cattle reference genomes. We propose trio binning as a new best practice for diploid genome assembly that will enable new studies of haplotype variation and inheritance.

biorxiv genomics 100-200-users 2018

Best Practices for Benchmarking Germline Small Variant Calls in Human Genomes, bioRxiv, 2018-02-24

AbstractAssessing accuracy of NGS variant calling is immensely facilitated by a robust benchmarking strategy and tools to carry it out in a standard way. Benchmarking variant calls requires careful attention to definitions of performance metrics, sophisticated comparison approaches, and stratification by variant type and genome context. The Global Alliance for Genomics and Health (GA4GH) Benchmarking Team has developed standardized performance metrics and tools for benchmarking germline small variant calls. This team includes representatives from sequencing technology developers, government agencies, academic bioinformatics researchers, clinical laboratories, and commercial technology and bioinformatics developers for whom benchmarking variant calls is essential to their work. Benchmarking variant calls is a challenging problem for many reasons<jatslist list-type=bullet><jatslist-item>Evaluating variant calls requires complex matching algorithms and standardized counting because the same variant may be represented differently in truth and query callsets.<jatslist-item><jatslist-item>Defining and interpreting resulting metrics such as precision (aka positive predictive value = TP(TP+FP)) and recall (aka sensitivity = TP(TP+FN)) requires standardization to draw robust conclusions about comparative performance for different variant calling methods.<jatslist-item><jatslist-item>Performance of NGS methods can vary depending on variant types and genome context; and as a result understanding performance requires meaningful stratification.<jatslist-item><jatslist-item>High-confidence variant calls and regions that can be used as “truth” to accurately identify false positives and negatives are difficult to define, and reliable calls for the most challenging regions and variants remain out of reach.<jatslist-item>We have made significant progress on standardizing comparison methods, metric definitions and reporting, as well as developing and using truth sets. Our methods are publicly available on GitHub (<jatsext-link xmlnsxlink=httpwww.w3.org1999xlink ext-link-type=uri xlinkhref=httpsgithub.comga4ghbenchmarking-tools>httpsgithub.comga4ghbenchmarking-tools<jatsext-link>) and in a web-based app on precisionFDA, which allow users to compare their variant calls against truth sets and to obtain a standardized report on their variant calling performance. Our methods have been piloted in the precisionFDA variant calling challenges to identify the best-in-class variant calling methods within high-confidence regions. Finally, we recommend a set of best practices for using our tools and critically evaluating the results.

biorxiv genomics 100-200-users 2018

Genetic meta-analysis identifies 9 novel loci and functional pathways for Alzheimer’s disease risk, bioRxiv, 2018-02-21

AbstractLate onset Alzheimer’s disease (AD) is the most common form of dementia with more than 35 million people affected worldwide, and no curative treatment available. AD is highly heritable and recent genome-wide meta-analyses have identified over 20 genomic loci associated with AD, yet only explaining a small proportion of the genetic variance indicating that undiscovered loci exist. Here, we performed the largest genome-wide association study of clinically diagnosed AD and AD-by-proxy (71,880 AD cases, 383,378 controls). AD-by-proxy status is based on parental AD diagnosis, and showed strong genetic correlation with AD (rg=0.81). Genetic meta analysis identified 29 risk loci, of which 9 are novel, and implicating 215 potential causative genes. Independent replication further supports these novel loci in AD. Associated genes are strongly expressed in immune-related tissues and cell types (spleen, liver and microglia). Furthermore, gene-set analyses indicate the genetic contribution of biological mechanisms involved in lipid-related processes and degradation of amyloid precursor proteins. We show strong genetic correlations with multiple health-related outcomes, and Mendelian randomisation results suggest a protective effect of cognitive ability on AD risk. These results are a step forward in identifying more of the genetic factors that contribute to AD risk and add novel insights into the neurobiology of AD to guide new drug development.

biorxiv genetics 100-200-users 2018

Understanding 6th-Century Barbarian Social Organization and Migration through Paleogenomics, bioRxiv, 2018-02-21

ABSTARCTDespite centuries of research, much about the barbarian migrations that took place between the fourth and sixth centuries in Europe remains hotly debated. To better understand this key era that marks the dawn of modern European societies, we obtained ancient genomic DNA from 63 samples from two cemeteries (from Hungary and Northern Italy) that have been previously associated with the Longobards, a barbarian people that ruled large parts of Italy for over 200 years after invading from Pannonia in 568 CE. Our dense cemetery-based sampling revealed that each cemetery was primarily organized around one large pedigree, suggesting that biological relationships played an important role in these early Medieval societies. Moreover, we identified genetic structure in each cemetery involving at least two groups with different ancestry that were very distinct in terms of their funerary customs. Finally, our data was consistent with the proposed long-distance migration from Pannonia to Northern Italy.

biorxiv genetics 100-200-users 2018