Genome-wide profiling of nucleosome position and chromatin accessibility in single cells using scMNase-seq

Due to the increasing throughput of current DNA sequencing instruments, sample multiplexing is necessary for making economical use of available sequencing capacities. A widely used multiplexing strategy for the Illumina Genome Analyzer utilizes sample-specific indexes, which are embedded in one of the library adapters. However, this and similar multiplex approaches come with a risk of sample misidentification. By introducing indexes into both library adapters (double indexing), we have developed a method that reveals the rate of sample misidentification within current multiplex sequencing experiments. With ~0.3% these rates are orders of magnitude higher than expected and may severely confound applications in cancer genomics and other fields requiring accurate detection of rare variants. We identified the occurrence of mixed clusters on the flow as the predominant source of error. The accuracy of sample identification is further impaired if indexed oligonucleotides are cross-contaminated or if indexed libraries are amplified in bulk. Double-indexing eliminates these problems and increases both the scope and accuracy of multiplex sequencing on the Illumina platform.

Nucleosome organization is critical for gene regulation. In living cells this organization is determined by multiple factors, including the action of chromatin remodellers, competition with site-specific DNA-binding proteins, and the DNA sequence preferences of the nucleosomes themselves. However, it has been difficult to estimate the relative importance of each of these mechanisms in vivo, because in vivo nucleosome maps reflect the combined action of all influencing factors. Here we determine the importance of nucleosome DNA sequence preferences experimentally by measuring the genome-wide occupancy of nucleosomes assembled on purified yeast genomic DNA. The resulting map, in which nucleosome occupancy is governed only by the intrinsic sequence preferences of nucleosomes, is similar to in vivo nucleosome maps generated in three different growth conditions. In vitro, nucleosome depletion is evident at many transcription factor binding sites and around gene start and end sites, indicating that nucleosome depletion at these sites in vivo is partly encoded in the genome. We confirm these results with a micrococcal nuclease-independent experiment that measures the relative affinity of nucleosomes for approximately 40,000 double-stranded 150-base-pair oligonucleotides. Using our in vitro data, we devise a computational model of nucleosome sequence preferences that is significantly correlated with in vivo nucleosome occupancy in Caenorhabditis elegans. Our results indicate that the intrinsic DNA sequence preferences of nucleosomes have a central role in determining the organization of nucleosomes in vivo.

Knowing the precise locations of nucleosomes in a genome is key to understanding how genes are regulated. Recent 'next generation' ChIP-chip and ChIP-Seq technologies have accelerated our understanding of the basic principles of chromatin organization. Here we discuss what high-resolution genome-wide maps of nucleosome positions have taught us about how nucleosome positioning demarcates promoter regions and transcriptional start sites, and how the composition and structure of promoter nucleosomes facilitate or inhibit transcription. A detailed picture is starting to emerge of how diverse factors, including underlying DNA sequences and chromatin remodelling complexes, influence nucleosome positioning.