Transcriptional and epigenetic dynamics during specification of human embryonic stem cells.

Differentiation of human embryonic stem cells (hESCs) provides a unique opportunity to study the regulatory mechanisms that facilitate cellular transitions in a human context. To that end, we performed comprehensive transcriptional and epigenetic profiling of populations derived through directed differentiation of hESCs representing each of the three embryonic germ layers. Integration of whole-genome bisulfite sequencing, chromatin immunoprecipitation sequencing, and RNA sequencing reveals unique events associated with specification toward each lineage. Lineage-specific dynamic alterations in DNA methylation and H3K4me1 are evident at putative distal regulatory elements that are frequently bound by pluripotency factors in the undifferentiated hESCs. In addition, we identified germ-layer-specific H3K27me3 enrichment at sites exhibiting high DNA methylation in the undifferentiated state. A better understanding of these initial specification events will facilitate identification of deficiencies in current approaches, leading to more faithful differentiation strategies as well as providing insights into the rewiring of human regulatory programs during cellular transitions.

Characterization and remediation of sample index swaps by non-redundant dual indexing on massively parallel sequencing platforms.

BACKGROUND: Here we present an in-depth characterization of the mechanism of sequencer-induced sample contamination due to the phenomenon of index swapping that impacts Illumina sequencers employing patterned flow cells with Exclusion Amplification (ExAmp) chemistry (HiSeqX, HiSeq4000, and NovaSeq). We also present a remediation method that minimizes the impact of such swaps. RESULTS: Leveraging data collected over a two-year period, we demonstrate the widespread prevalence of index swapping in patterned flow cell data. We calculate mean swap rates across multiple sample preparation methods and sequencer models, demonstrating that different library methods can have vastly different swapping rates and that even non-ExAmp chemistry instruments display trace levels of index swapping. We provide methods for eliminating sample data cross contamination by utilizing non-redundant dual indexing for complete filtering of index swapped reads, and share the sequences for 96 non-combinatorial dual indexes we have validated across various library preparation methods and sequencer models. Finally, using computational methods we provide a greater insight into the mechanism of index swapping. CONCLUSIONS: Index swapping in pooled libraries is a prevalent phenomenon that we observe at a rate of 0.2 to 6% in all sequencing runs on HiSeqX, HiSeq 4000/3000, and NovaSeq. Utilizing non-redundant dual indexing allows for the removal (flagging/filtering) of these swapped reads and eliminates swapping induced sample contamination, which is critical for sensitive applications such as RNA-seq, single cell, blood biopsy using circulating tumor DNA, or clinical sequencing.

Comprehensive variation discovery in single human genomes.

Complete knowledge of the genetic variation in individual human genomes is a crucial foundation for understanding the etiology of disease. Genetic variation is typically characterized by sequencing individual genomes and comparing reads to a reference. Existing methods do an excellent job of detecting variants in approximately 90% of the human genome; however, calling variants in the remaining 10% of the genome (largely low-complexity sequence and segmental duplications) is challenging. To improve variant calling, we developed a new algorithm, DISCOVAR, and examined its performance on improved, low-cost sequence data. Using a newly created reference set of variants from the finished sequence of 103 randomly chosen fosmids, we find that some standard variant call sets miss up to 25% of variants. We show that the combination of new methods and improved data increases sensitivity by several fold, with the greatest impact in challenging regions of the human genome.

Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling.

Sequencing-based approaches have led to new insights about DNA methylation. While many different techniques for genome-scale mapping of DNA methylation have been employed, throughput has been a key limitation for most. To further facilitate the mapping of DNA methylation, we describe a protocol for gel-free multiplexed reduced representation bisulfite sequencing (mRRBS) that reduces the workload dramatically and enables processing of 96 or more samples per week. mRRBS achieves similar CpG coverage to the original RRBS protocol, while the higher throughput and lower cost make it better suited for large-scale DNA methylation mapping studies, including cohorts of cancer samples.