Behind the scenes: Science that drives Illumina\u27s sequencing chemistry

DNA polymerase is the engine of Illumina’s Sequencing by Synthesis (SBS) technology. In the past few years, we have invented and adopted an array of cross-disciplinary approaches to understand the fundamental mechanics of the polymerase and how it interacts with the rest of the sequencing system. This work has led to several generations of polymerase variants that enabled significant improvements of the sequencing metrics, including turnaround time, read length and accuracy. This will be the first time we share some of the fundamental science behind the chemistry improvements with the scientific community. We present structural, biophysical and kinetic studies to understand the relationship between the polymerase’s molecular properties and the sequencing metrics. Based on the understanding of the mechanism, high throughput solution assays are developed to predict the mutant performances on sequencers. By a combination of screening methods, random and targeted mutant libraries are made and assayed to discover faster and better polymerases that drive the ever improving Illumina sequencing chemistry. We use a similar scientific approach to understand and evolve the library prep enzyme transposase that is the core of the Nextera® library preparation kits. We present a short overview of how protein engineering and machine learning approaches are utilized to improve the insertion bias of the Nextera® transposase. We end with future directions to further advance Illumina’s sequencing technology by protein engineering technologies. We also discuss the possibilities of using Illumina sequencers as a novel high throughput imaging tool to advance biological research for a wider scientific community

Characterization of chromatin accessibility with a transposome hypersensitive sites sequencing (THS-seq) assay.

Chromatin accessibility captures in vivo protein-chromosome binding status, and is considered an informative proxy for protein-DNA interactions. DNase I and Tn5 transposase assays require thousands to millions of fresh cells for comprehensive chromatin mapping. Applying Tn5 tagmentation to hundreds of cells results in sparse chromatin maps. We present a transposome hypersensitive sites sequencing assay for highly sensitive characterization of chromatin accessibility. Linear amplification of accessible DNA ends with in vitro transcription, coupled with an engineered Tn5 super-mutant, demonstrates improved sensitivity on limited input materials, and accessibility of small regions near distal enhancers, compared with ATAC-seq

A systematic review of the prevalence of lifetime experience with 'conversion' practices among sexual and gender minority populations.

RationaleConversion practices (CPs) refer to organized attempts to deter people from adopting or expressing non-heterosexual identities or gender identities that differ from their gender/sex assigned at birth. Numerous jurisdictions have contemplated or enacted legislative CP bans in recent years. Syntheses of CP prevalence are needed to inform further public health policy and action.ObjectivesTo conduct a systematic review describing CP prevalence estimates internationally and exploring heterogeneity across country and socially relevant subgroups.MethodsWe performed literature searches in eight databases (Medline, Embase, PsycInfo, Social Work Abstracts, CINAHL, Web of Science, LGBTQ+ Source, and Proquest Dissertations) and included studies from all jurisdictions, globally, conducted after 2000 with a sampling frame of sexual and gender minority (SGM) people, as well as studies of practitioners seeing SGM patients. We used the Hoy et al. risk of bias tool for prevalence studies and summarized distribution of estimates using median and range.ResultsWe identified fourteen articles that reported prevalence estimates among SGM populations, and two articles that reported prevalence estimates from studies of mental health practitioners. Prevalence estimates among SGM samples ranged 2%-34% (median: 8.5). Prevalence estimates were greater in studies conducted in the US (median: 13%), compared to Canada (median: 7%), and greater among transgender (median: 12%), compared to cisgender (median: 4%) subsamples. Prevalence estimates were greatest among people assigned male at birth, whether transgender (median: 10%) or cisgender (median: 8%), as compared to people assigned female at birth (medians: 5% among transgender participants, 3% among cisgender participants). Further differences were observed by race (medians: 8% among Indigenous and other racial minorities, 5% among white groups) but not by sexual orientation.ConclusionsCPs remain prevalent, despite denouncements from professional bodies. Social inequities in CP prevalence signal the need for targeted efforts to protect transgender, Indigenous and racial minority, and assigned-male-at-birth subgroups

Additional file 1: Figure S1. of Characterization of chromatin accessibility with a transposome hypersensitive sites sequencing (THS-seq) assay

Schematic of transposon design and transposome complex generation. Figure S2. Validation of successful transposon insertion and DNA fragmentation. Figure S3. Validation of T7 in vitro transcription. Figure S4. Validation of PCR amplification for barcode addition. Figure S5. Tn5059 concentration and dimethylformamide titrations for optimal in vitro transcription amplification yields. Figure S6. Comparison of peak region distances to transcription start sites. Figure S7. Significant gene ontology biological processes categories for each peaks dataset. Figure S8. Quantitation of mitochondrial reads in all datasets. Figure S9. 100-cell THS-seq/Tn5059 read statistics analysis. Figure S10. Peak overlap comparisons in all datasets. Figure S11. Base pair overlap comparisons in all datasets. Figure S12. Examination of peak overlap in a pairwise comparison of all experimental datasets. Figure S13. Examination of base pair overlap in a pairwise comparison of all experimental datasets. Figure S14. Two hundred kilobyte view of accessible chromatin marks in 500-cell datasets. Figure S15. Overview of chromatin accessibility at gene loci enriched in 100- and 500-cell THS-seq/Tn5059 data and not enriched in 500-cell ATAC-seq/EzTn5 data. Figure S16. Overview of chromatin accessibility at major genes implicated in cancer and in immune system function. Figure S17. Peak size distribution comparisons between THS-seq, ATAC-seq, and ENCODE data. Figure S18. Validation of peaks based on peak length. Figure S19. THS-seq and ATAC-seq peak capture preferences. Figure S20. Comparison of 500-cell ATAC-seq/EzTn5 data to published ATAC-seq data. Figure S21. Two hundred kilobyte view of accessible chromatin in ATAC-seq/EzTn5 data versus published ATAC-seq data. Table S1. 100-cell THS-seq/Tn5059 data and controls with read statistics and data analysis. Table S2. Peak size counts versus lengths of Dfilter called peaks for each sample. Table S3. Analysis of datasets down-sampled to 8,351,125 unique reads and analysis of published ATAC-seq datasets. Table S4. Original datasets analysis results. Table S5. THS-seq oligo sequences

Additional file 4: Figure S3. of Improved genome sequencing using an engineered transposase

Schematic representation of Tn5 tagmentation. There is 9 base pairs overlap between top and bottom strands. Two Tn5 transposase monomers, Tn5-A and Tn5-B form a dimer and tagment a double stranded DNA. Forward and reverse strands are shown in the figure. A typical bias plot from sequencing of B. cereus is also shown in the figure (refer to Fig. 3a). For every base at position P (where P is between 1 and 9) in a read, theoretically there is another read in the sequencing results that has complementary nucleotide to that base at position 10-P. This results in a symmetry between positions 1 and 9 in the bias plots, and the center of the symmetry will be at position 5. (PDF 113 kb

Additional file 2: Figure S1. of Improved genome sequencing using an engineered transposase

Normalization of tagmentation activity based on pre-PCR insertion size distribution. Activity is normalized when 20–30% of the library has an insert size of 100–300 bp, 20–30% of 301–600 bp, and at least 90% of the library falls in the range of 100 bp-7000 bp. TDE1 refers to standard Tn5 Transposme complex that comes in Nextera kit. Here, TDE1 represents standard tagmentation using Nextera kit. (PDF 54 kb

Additional file 3: Figure S2. of Improved genome sequencing using an engineered transposase

DNA binding stability of Tn5 vs. Tn5-059. While heating at 74.2 °C is required for Tn5 to dissociate from tagmented DNA to allow following PCR amplification of DNA to reach the level of positive control, the temperature is elevated to 76.6 °C for Tn5-059 to do the same. The negative control is tagmentation without PCR amplification. The positive control is tagmentation followed by complete removal of transposase by Zymo cleaning to allow PCR amplification. (PDF 81 kb

Additional file 6: Figure S5. of Improved genome sequencing using an engineered transposase

Linear regression weights on selected amino acids positions. This plot shows the different effect of a mutation on the insertion bias at different positions. (PDF 58 kb