Pacybara: accurate long-read sequencing for barcoded mutagenized allelic libraries.

MOTIVATION: Long-read sequencing technologies, an attractive solution for many applications, often suffer from higher error rates. Alignment of multiple reads can improve base-calling accuracy, but some applications, e.g. sequencing mutagenized libraries where multiple distinct clones differ by one or few variants, require the use of barcodes or unique molecular identifiers. Unfortunately, sequencing errors can interfere with correct barcode identification, and a given barcode sequence may be linked to multiple independent clones within a given library. RESULTS: Here we focus on the target application of sequencing mutagenized libraries in the context of multiplexed assays of variant effects (MAVEs). MAVEs are increasingly used to create comprehensive genotype-phenotype maps that can aid clinical variant interpretation. Many MAVE methods use long-read sequencing of barcoded mutant libraries for accurate association of barcode with genotype. Existing long-read sequencing pipelines do not account for inaccurate sequencing or nonunique barcodes. Here, we describe Pacybara, which handles these issues by clustering long reads based on the similarities of (error-prone) barcodes while also detecting barcodes that have been associated with multiple genotypes. Pacybara also detects recombinant (chimeric) clones and reduces false positive indel calls. In three example applications, we show that Pacybara identifies and correctly resolves these issues. AVAILABILITY AND IMPLEMENTATION: Pacybara, freely available at https://github.com/rothlab/pacybara, is implemented using R, Python, and bash for Linux. It runs on GNU/Linux HPC clusters via Slurm, PBS, or GridEngine schedulers. A single-machine simplex version is also available.

Pacybara: Accurate long-read sequencing for barcoded mutagenized allelic libraries.

Long read sequencing technologies, an attractive solution for many applications, often suffer from higher error rates. Alignment of multiple reads can improve base-calling accuracy, but some applications, e.g. sequencing mutagenized libraries where multiple distinct clones differ by one or few variants, require the use of barcodes or unique molecular identifiers. Unfortunately, sequencing errors can interfere with correct barcode identification, and a given barcode sequence may be linked to multiple independent clones within a given library. Here we focus on the target application of sequencing mutagenized libraries in the context of multiplexed assays of variant effects (MAVEs). MAVEs are increasingly used to create comprehensive genotype-phenotype maps that can aid clinical variant interpretation. Many MAVE methods use long-read sequencing of barcoded mutant libraries for accurate association of barcode with genotype. Existing long-read sequencing pipelines do not account for inaccurate sequencing or non-unique barcodes. Here, we describe Pacybara, which handles these issues by clustering long reads based on the similarities of (error-prone) barcodes while also detecting barcodes that have been associated with multiple genotypes. Pacybara also detects recombinant (chimeric) clones and reduces false positive indel calls. In three example applications, we show that Pacybara identifies and correctly resolves these issues.

SNCA genetic lowering reveals differential cognitive function of alpha-synuclein dependent on sex.

Antisense oligonucleotide (ASO) therapy for neurological disease has been successful in clinical settings and its potential has generated hope for Alzheimer's disease (AD). We previously described that ablating SNCA encoding for α-synuclein (αSyn) in a mouse model of AD was beneficial. Here, we sought to demonstrate whether transient reduction of αSyn expression using ASOSNCA could be therapeutic in a mouse model of AD. The efficacy of the ASOSNCA was measured via immunocytochemistry, RT-qPCR and western blotting. To assess spatial learning and memory, ASOSNCA or PBS-injected APP and non-transgenic (NTG) mice, and separate groups of SNCA-null mice, were tested on the Barnes circular maze. Hippocampal slice electrophysiology and transcriptomic profiling were used to explore synaptic function and differential gene expression between groups. Reduction of SNCA transcripts alleviated cognitive deficits in male transgenic animals, but surprisingly, not in females. To determine the functional cause of this differential effect, we assessed memory function in SNCA-null mice. Learning and memory were intact in male mice but impaired in female animals, revealing that the role of αSyn on cognitive function is sex-specific. Transcriptional analyses identified a differentially expressed gene network centered around EGR1, a central modulator of learning and memory, in the hippocampi of SNCA-null mice. Thus, these novel results demonstrate that the function of αSyn on memory differs between male and female brains.

Measuring Pharmacogene Variant Function at Scale Using Multiplexed Assays.

As costs of next-generation sequencing decrease, identification of genetic variants has far outpaced our ability to understand their functional consequences. This lack of understanding is a central challenge to a key promise of pharmacogenomics: using genetic information to guide drug selection and dosing. Recently developed multiplexed assays of variant effect enable experimental measurement of the function of thousands of variants simultaneously. Here, we describe multiplexed assays that have been performed on nearly 25,000 variants in eight key pharmacogenes (ADRB2, CYP2C9, CYP2C19, NUDT15, SLCO1B1, TMPT, VKORC1, and the LDLR promoter), discuss advances in experimental design, and explore key challenges that must be overcome to maximize the utility of multiplexed functional data.

Massively parallel characterization of CYP2C9 variant enzyme activity and abundance.

CYP2C9 encodes a cytochrome P450 enzyme responsible for metabolizing up to 15% of small molecule drugs, and CYP2C9 variants can alter the safety and efficacy of these therapeutics. In particular, the anti-coagulant warfarin is prescribed to over 15 million people annually and polymorphisms in CYP2C9 can affect individual drug response and lead to an increased risk of hemorrhage. We developed click-seq, a pooled yeast-based activity assay, to test thousands of variants. Using click-seq, we measured the activity of 6,142 missense variants in yeast. We also measured the steady-state cellular abundance of 6,370 missense variants in a human cell line by using variant abundance by massively parallel sequencing (VAMP-seq). These data revealed that almost two-thirds of CYP2C9 variants showed decreased activity and that protein abundance accounted for half of the variation in CYP2C9 function. We also measured activity scores for 319 previously unannotated human variants, many of which may have clinical relevance.

Bidirectional modulation of Alzheimer phenotype by alpha-synuclein in mice and primary neurons.

α-Synuclein (αSyn) histopathology defines several neurodegenerative disorders, including Parkinson's disease, Lewy body dementia, and Alzheimer's disease (AD). However, the functional link between soluble αSyn and disease etiology remains elusive, especially in AD. We, therefore, genetically targeted αSyn in APP transgenic mice modeling AD and mouse primary neurons. Our results demonstrate bidirectional modulation of behavioral deficits and pathophysiology by αSyn. Overexpression of human wild-type αSyn in APP animals markedly reduced amyloid deposition but, counter-intuitively, exacerbated deficits in spatial memory. It also increased extracellular amyloid-ß oligomers (AßOs), αSyn oligomers, exacerbated tau conformational and phosphorylation variants associated with AD, and enhanced neuronal cell cycle re-entry (CCR), a frequent prelude to neuron death in AD. Conversely, ablation of the SNCA gene encoding for αSyn in APP mice improved memory retention in spite of increased plaque burden. Reminiscent of the effect of MAPT ablation in APP mice, SNCA deletion prevented premature mortality. Moreover, the absence of αSyn decreased extracellular AßOs, ameliorated CCR, and rescued postsynaptic marker deficits. In summary, this complementary, bidirectional genetic approach implicates αSyn as an essential mediator of key phenotypes in AD and offers new functional insight into αSyn pathophysiology.

The amyloid-ß oligomer Aß*56 induces specific alterations in neuronal signaling that lead to tau phosphorylation and aggregation.

Oligomeric forms of amyloid-forming proteins are believed to be the principal initiating bioactive species in many neurodegenerative disorders, including Alzheimer's disease (AD). Amyloid-ß (Aß) oligomers are implicated in AD-associated phosphorylation and aggregation of the microtubule-associated protein tau. To investigate the specific molecular pathways activated by different assemblies, we isolated various forms of Aß from Tg2576 mice, which are a model for AD. We found that Aß*56, a 56-kDa oligomer that is detected before patients develop overt signs of AD, induced specific changes in neuronal signaling. In primary cortical neurons, Aß*56 interacted with N-methyl-d-aspartate receptors (NMDARs), increased NMDAR-dependent Ca2+ influx, and consequently increased intracellular calcium concentrations and the activation of Ca2+-dependent calmodulin kinase IIα (CaMKIIα). In cultured neurons and in the brains of Tg2576 mice, activated CaMKIIα was associated with increased site-specific phosphorylation and missorting of tau, both of which are associated with AD pathology. In contrast, exposure of cultured primary cortical neurons to other oligomeric Aß forms (dimers and trimers) did not trigger these effects. Our results indicate that distinct Aß assemblies activate neuronal signaling pathways in a selective manner and that dissecting the molecular events caused by each oligomer may inform more effective therapeutic strategies.

Selective lowering of synapsins induced by oligomeric α-synuclein exacerbates memory deficits.

Mounting evidence indicates that soluble oligomeric forms of amyloid proteins linked to neurodegenerative disorders, such as amyloid-ß (Aß), tau, or α-synuclein (αSyn) might be the major deleterious species for neuronal function in these diseases. Here, we found an abnormal accumulation of oligomeric αSyn species in AD brains by custom ELISA, size-exclusion chromatography, and nondenaturing/denaturing immunoblotting techniques. Importantly, the abundance of αSyn oligomers in human brain tissue correlated with cognitive impairment and reductions in synapsin expression. By overexpressing WT human αSyn in an AD mouse model, we artificially enhanced αSyn oligomerization. These bigenic mice displayed exacerbated Aß-induced cognitive deficits and a selective decrease in synapsins. Following isolation of various soluble αSyn assemblies from transgenic mice, we found that in vitro delivery of exogenous oligomeric αSyn but not monomeric αSyn was causing a lowering in synapsin-I/II protein abundance. For a particular αSyn oligomer, these changes were either dependent or independent on endogenous αSyn expression. Finally, at a molecular level, the expression of synapsin genes SYN1 and SYN2 was down-regulated in vivo and in vitro by αSyn oligomers, which decreased two transcription factors, cAMP response element binding and Nurr1, controlling synapsin gene promoter activity. Overall, our results demonstrate that endogenous αSyn oligomers can impair memory by selectively lowering synapsin expression.