ERASE-Seq: Leveraging replicate measurements to enhance ultralow frequency variant detection in NGS data.

The accurate detection of ultralow allele frequency variants in DNA samples is of interest in both research and medical settings, particularly in liquid biopsies where cancer mutational status is monitored from circulating DNA. Next-generation sequencing (NGS) technologies employing molecular barcoding have shown promise but significant sensitivity and specificity improvements are still needed to detect mutations in a majority of patients before the metastatic stage. To address this we present analytical validation data for ERASE-Seq (Elimination of Recurrent Artifacts and Stochastic Errors), a method for accurate and sensitive detection of ultralow frequency DNA variants in NGS data. ERASE-Seq differs from previous methods by creating a robust statistical framework to utilize technical replicates in conjunction with background error modeling, providing a 10 to 100-fold reduction in false positive rates compared to published molecular barcoding methods. ERASE-Seq was tested using spiked human DNA mixtures with clinically realistic DNA input quantities to detect SNVs and indels between 0.05% and 1% allele frequency, the range commonly found in liquid biopsy samples. Variants were detected with greater than 90% sensitivity and a false positive rate below 0.1 calls per 10,000 possible variants. The approach represents a significant performance improvement compared to molecular barcoding methods and does not require changing molecular reagents.

A comparative study of ChIP-seq sequencing library preparation methods.

BACKGROUND: ChIP-seq is the primary technique used to investigate genome-wide protein-DNA interactions. As part of this procedure, immunoprecipitated DNA must undergo "library preparation" to enable subsequent high-throughput sequencing. To facilitate the analysis of biopsy samples and rare cell populations, there has been a recent proliferation of methods allowing sequencing library preparation from low-input DNA amounts. However, little information exists on the relative merits, performance, comparability and biases inherent to these procedures. Notably, recently developed single-cell ChIP procedures employing microfluidics must also employ library preparation reagents to allow downstream sequencing. RESULTS: In this study, seven methods designed for low-input DNA/ChIP-seq sample preparation (Accel-NGS® 2S, Bowman-method, HTML-PCR, SeqPlex™, DNA SMART™, TELP and ThruPLEX®) were performed on five replicates of 1 ng and 0.1 ng input H3K4me3 ChIP material, and compared to a "gold standard" reference PCR-free dataset. The performance of each method was examined for the prevalence of unmappable reads, amplification-derived duplicate reads, reproducibility, and for the sensitivity and specificity of peak calling. CONCLUSIONS: We identified consistent high performance in a subset of the tested reagents, which should aid researchers in choosing the most appropriate reagents for their studies. Furthermore, we expect this work to drive future advances by identifying and encouraging use of the most promising methods and reagents. The results may also aid judgements on how comparable are existing datasets that have been prepared with different sample library preparation reagents.

Guiding the morphogenesis of dissociated newborn mouse retinal cells and hES cell-derived retinal cells by soft lithography-patterned microchannel PLGA scaffolds.

Embryonic stem (ES) cell-derived photoreceptors are a promising cell source for enhanced in vitro models of retinal degenerative diseases, but the more differentiated characteristics of retinal cells do not typically develop in dissociated cell cultures. Therefore, we have reconstructed organized retinal tissue by seeding dissociated cells into an array of aligned units that more faithfully mimics the retina. We solvent-processed poly(lactic-co-glycolic acid) (PLGA) into a microchannel scaffold format to achieve this geometric constraint. We compared the effect of PLGA concentration on channel morphology and, along with other culture conditions, on the infiltration of dissociated newborn mouse retinal cells into the channels. Culturing scaffolds at the gas-liquid interface with low serum media increased infiltrated rod photoreceptor viability 18-fold over submerged, high serum cultures when evaluated after seven days. Rod photoreceptors and Müller glia aligned processes parallel to the microchannel walls. Otx2+ and Pax6+ subpopulations recapitulated lamination behavior. Further, we constructed scaffold/retinal pigment epithelium (RPE) co-cultures and observed rods extending rhodopsin-positive processes toward RPE cells, mimicking normal rod polarization and morphology. Finally, human embryonic stem cell-derived photoreceptors exhibited infiltration and morphological characteristics similar to mouse retinal cells inside the scaffolds. These findings constitute an important advance in generating tissue-level retinal models from dissociated cells for use as drug screening platforms and in regenerative medicine.

Generation, purification and transplantation of photoreceptors derived from human induced pluripotent stem cells.

BACKGROUND: Inherited and acquired retinal degenerations are frequent causes of visual impairment and photoreceptor cell replacement therapy may restore visual function to these individuals. To provide a source of new retinal neurons for cell based therapies, we developed methods to derive retinal progenitors from human ES cells. METHODOLOGY/PHYSICAL FINDINGS: In this report we have used a similar method to direct induced pluripotent stem cells (iPS) from human fibroblasts to a retinal progenitor fate, competent to generate photoreceptors. We also found we could purify the photoreceptors derived from the iPS cells using fluorescence activated cell sorting (FACS) after labeling photoreceptors with a lentivirus driving GFP from the IRBP cis-regulatory sequences. Moreover, we found that when we transplanted the FACS purified iPSC derived photoreceptors, they were able to integrate into a normal mouse retina and express photoreceptor markers. CONCLUSIONS: This report provides evidence that enriched populations of human photoreceptors can be derived from iPS cells.