Estimating Exceptionally Rare Germline and Somatic Mutation Frequencies via Next Generation Sequencing.

We used targeted next generation deep-sequencing (Safe Sequencing System) to measure ultra-rare de novo mutation frequencies in the human male germline by attaching a unique identifier code to each target DNA molecule. Segments from three different human genes (FGFR3, MECP2 and PTPN11) were studied. Regardless of the gene segment, the particular testis donor or the 73 different testis pieces used, the frequencies for any one of the six different mutation types were consistent. Averaging over the C>T/G>A and G>T/C>A mutation types the background mutation frequency was 2.6x10-5 per base pair, while for the four other mutation types the average background frequency was lower at 1.5x10-6 per base pair. These rates far exceed the well documented human genome average frequency per base pair (~10-8) suggesting a non-biological explanation for our data. By computational modeling and a new experimental procedure to distinguish between pre-mutagenic lesion base mismatches and a fully mutated base pair in the original DNA molecule, we argue that most of the base-dependent variation in background frequency is due to a mixture of deamination and oxidation during the first two PCR cycles. Finally, we looked at a previously studied disease mutation in the PTPN11 gene and could easily distinguish true mutations from the SSS background. We also discuss the limits and possibilities of this and other methods to measure exceptionally rare mutation frequencies, and we present calculations for other scientists seeking to design their own such experiments

Read and UID family statistics for the three experiments.

<p>Read and UID family statistics for the three experiments.</p

Mutation frequency as a function of mutation type.

<p>Mutation frequency as a function of mutation type.</p

Second Round of the SSS Strategy.

<p><b>a.</b> Universal forward and reverse primers are used to amplify the product made in the first round of PCR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158340#pone.0158340.g001" target="_blank">Fig 1E</a>). The primers contain the Illumina Sequencing primer (ISP), which is partially complementary to the Sequencing primer region of the primers used in the first two PCR cycles (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158340#pone.0158340.g001" target="_blank">Fig 1A</a>). The universal primers also contain the complement of the Flow cell grafting sequence (FC). <b>b</b>. Products of the first few cycles of the second round using the template shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158340#pone.0158340.g001" target="_blank">Fig 1E</a>. <b>c</b>. Further amplification for 28–30 additional cycles leads to the creation of UID families of which the one shown is representative. The original genomic mutation and the mutation that arose during the first two SSS PCR cycles are both present in this particular family. Notice that one of the family members has accumulated an additional <i>in vitro</i> mutation (black star) due to events during the second PCR round or the final sequencing step. <b>d.</b> Analysis of the UID family is able to eliminate the latter error from consideration.</p

Spurious super-mutants due to very large UID families.

<p>One very large UID family on the top row is erroneously counted as twelve additional families. The next nine rows show families with UID sequences that differ at one site from the family in the top row, most likely due to a mistake during PCR amplification. The bottom three rows show families with the same long UID sequence but a different short UID sequence from the family in the top row, most likely due to PCR jumping. All of these families contain the same A>T/T>A super-mutant at the read position 10 bases from the end of the UID (p.10A>T) erroneously increasing its frequency.</p

<i>PTPN11</i> mutation frequencies color-coded by reference base.

<p>Each dot represents the average of 9 different libraries from three testis pieces. Red indicates an A or T base, blue indicates a C or G (non-CpG) base, and green indicates a CpG. The mutation frequency is the sum of all mutations at that site, so, e.g., if a site is a C, the mutation frequency is the sum of the C>A, C>G, and C>T frequencies at that site. The 95% confidence interval for each position is also shown. The data on one base pair (at position 80; c.922) has not been included since it has a much greater mutation frequency; for an explanation, see below.</p

Separate method and pre-existing mismatches.

<p>The Separate method separately amplifies the coding and non-coding strands. The aliquot on the left-hand side only amplifies the top strand, while the aliquot on the right-hand side only amplifies the bottom strand. The yellow rectangles indicate strands that are not primer extension products and are removed by exonucleases. For the original molecule shown with a pre-existing U:G mismatch, the Separate method detects a C>T mutation on the left-hand side and no mutation on the right-hand side.</p

Mutation frequencies estimated by the Separate method.

<p>Mutation frequencies estimated by the Separate method.</p

Estimates of the PCR error rate for the different mutation types.

<p>Estimates of the PCR error rate for the different mutation types.</p

Reducing wait times and avoiding unnecessary use of high-cost mental health services through a Rapid Access and Stabilization Program: protocol for a program evaluation study

Abstract Background Emergency psychiatric care, unplanned hospital admissions, and inpatient health care are the costliest forms of mental health care. According to Statistics Canada (2018), almost 18% (5.3 million) of Canadians reported needing mental health support. However, just above half of this figure (56.2%) have reported their needs were fully met. In light of this evidence there is a pressing need to provide accessible mental health services in flexible yet cost-effective ways. To further expand capacity and access to mental health care in the province, Nova Scotia Health has launched a novel mental health initiative for people in need of mental health care without requiring emergency department visits or hospitalization. This new service is referred to as the Rapid Access and Stabilization Program (RASP). This study evaluates the effectiveness and impact of the RASP on high-cost health services utilization (e.g. ED visits, mobile crisis visits, and inpatient treatments) and related costs. It also assesses healthcare partners' (e.g. healthcare providers, policymakers, community leaders) perceptions and patient experiences and satisfaction with the program and identifies sociodemographic characteristics, psychological conditions, recovery, well-being, and risk measures in the assisted population. Method This is a hypothesis-driven program evaluation study that employs a mixed methods approach. A within-subject comparison (pre- and post-evaluation study) will examine health services utilization data from patients attending RASP, one year before and one year after their psychiatry assessment at the program. A controlled between-subject comparison (cohort study) will use historical data from a control population will examine whether possible changes in high-cost health services utilization are associated with the intervention (RASP). The primary analysis involves extracting secondary data from provincial information systems, electronic medical records, and regular self-reported clinical assessments. Additionally, a qualitative sub-study will examine patient experience and satisfaction, and health care partners' impressions. Discussion We expect that RASP evaluation findings will demonstrate a minimum 10% reduction in high-cost health services utilization and corresponding 10% cost savings, and also a reduction in the wait times for patient consultations with psychiatrists to less than 30 calendar days, in both within-subject and between-subject comparisons. In addition, we anticipate that patients, healthcare providers and healthcare partners would express high levels of satisfaction with the new service. Conclusion This study will demonstrate the results of the Mental Health and Addictions Program (MHAP) efforts to provide stepped-care, particularly community-based support, to individuals with mental illnesses. Results will provide new insights into a novel community-based approach to mental health service delivery and contribute to knowledge on how to implement mental health programs across varying contexts