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Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5′ end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

Molecular ecologists frequently use genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from many individuals (e.g., RADseq, GBS). However, researchers often find the existing methods expensive to initiate and/or difficult to implement consistently, especially because it is difficult to multiplex sufficient numbers of samples to fill entire sequencing lanes. Here, we introduce a low-cost and highly robust approach for the construction of dual-digest RADseq libraries that build on adapters and primers designed in Adapterama I. Major features of our method include: (1) minimizing the number of processing steps; (2) focusing on a single strand of sample DNA for library construction, allowing the use of a non-phosphorylated adapter on one end; (3) ligating adapters in the presence of active restriction enzymes, thereby reducing chimeras; (4) including an optional third restriction enzyme to cut apart adapter-dimers formed by the phosphorylated adapter, thus increasing the efficiency of adapter ligation to sample DNA, which is particularly effective when only low quantity/quality DNA samples are available; (5) interchangeable adapter designs; (6) incorporating variable-length internal indexes within the adapters to increase the scope of sample indexing, facilitate pooling, and increase sequence diversity; (7) maintaining compatibility with universal dual-indexed primers and thus, Illumina sequencing reagents and libraries; and, (8) easy modification for the identification of PCR duplicates. We present eight adapter designs that work with 72 restriction enzyme combinations. We demonstrate the efficiency of our approach by comparing it with existing methods, and we validate its utility through the discovery of many variable loci in a variety of non-model organisms. Our 2RAD/3RAD method is easy to perform, has low startup costs, has increased utility with low-concentration input DNA, and produces libraries that can be highly-multiplexed and pooled with other Illumina libraries.

Massively parallel DNA sequencing offers many benefits, but major inhibitory cost factors include: (1) start-up (i.e., purchasing initial reagents and equipment); (2) buy-in (i.e., getting the smallest possible amount of data from a run); and (3) sample preparation. Reducing sample preparation costs is commonly addressed, but start-up and buy-in costs are rarely addressed. We present dual-indexing systems to address all three of these issues. By breaking the library construction process into universal, re-usable, combinatorial components, we reduce all costs, while increasing the number of samples and the variety of library types that can be combined within runs. We accomplish this by extending the Illumina TruSeq dual-indexing approach to 768 (384 + 384) indexed primers that produce 384 unique dual-indexes or 147,456 (384 × 384) unique combinations. We maintain eight nucleotide indexes, with many that are compatible with Illumina index sequences. We synthesized these indexing primers, purifying them with only standard desalting and placing small aliquots in replicate plates. In qPCR validation tests, 206 of 208 primers tested passed (99% success). We then created hundreds of libraries in various scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter that works with indexed PCR primers to uniquely identify samples. Our approach reduces buy-in costs because: (1) relatively few oligonucleotides are needed to produce a large number of indexed libraries; and (2) the large number of possible primers allows researchers to use unique primer sets for different projects, which facilitates pooling of samples during sequencing. Our libraries make use of standard Illumina sequencing primers and index sequence length and are demultiplexed with standard Illumina software, thereby minimizing customization headaches. In subsequent Adapterama papers, we use these same primers with different adapter stubs to construct amplicon and restriction-site associated DNA libraries, but their use can be expanded to any type of library sequenced on Illumina platforms.

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5' end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

The article presents math activity programs for students in kindergarten through sixth grade which teach concepts about multiplication, addition, and subtraction using word problems about the Walt Disney Company's Disney amusement parks.

Background: To investigate the short-term effects of cyclopentolate and tropicamide eyedrops on choroidal thickness (ChT) in myopic children using placebo or low-dose atropine eyedrops., Methods: The analysis included 242 myopic individuals (7-19 years) enrolled in two randomised placebo-controlled clinical trials of low-dose atropine eyedrops. Cycloplegia was induced using either one drop of 1% cyclopentolate (n = 161), two drops of 1% cyclopentolate (n = 32) or two drops of 1% tropicamide (n = 49). ChT measurements were taken using swept-source optical coherence tomography before and 30 min after administering the cycloplegic eye drops. A subset of 51 participants underwent test-retest measurements prior to cycloplegia., Results: Mean changes in subfoveal ChT after two drops of tropicamide and one and two drops of cyclopentolate were -2.5 μm (p = 0.10), -4.3 μm (p < 0.001) and -9.6 μm (p < 0.001), respectively. Subfoveal ChT changes after one and two drops of cyclopentolate were significantly greater than the test-retest changes (test-retest mean change: -3.1 μm; p < 0.05), while the tropicamide group was not significantly different (p = 0.64). Choroidal thinning post-cyclopentolate was not significantly different between atropine and placebo treatment groups (p > 0.05 for all macular locations). The coefficient of repeatability (CoR) in the tropicamide group (range: 8.2-14.4 μm) was similar to test-retest (range: 7.5-12.2 μm), whereas greater CoR values were observed in the cyclopentolate groups (one drop: range: 10.8-15.3 μm; two drops: range: 12.2-24.6 μm)., Conclusions: Cyclopentolate eye drops caused dose-dependent choroidal thinning and increased variation in pre- to post-cycloplegia measurements compared with test-retest variability, whereas tropicamide did not. These findings have practical implications for ChT measurements when cyclopentolate is used, particularly for successive measurements., (© 2023 The Authors. Ophthalmic and Physiological Optics published by John Wiley & Sons Ltd on behalf of College of Optometrists.)

Introduction: The 21-gene assay provides prognostication for estrogen receptor positive, human epidermal growth factor receptor-2 negative (ER+/HER2-) early female breast cancer patients. This signature has not been validated in male breast cancer (MBC)., Methods: A systematic review and meta-analysis was performed in accordance to the PRISMA guidelines. Retrospective cohort studies comparing 21-gene assay scores in female and MBC were included. Dichotomous variables were pooled as odds ratios (OR) and associated 95% confidence intervals (CI) using the Mantel-Haenszel method., Results: Six studies including 176,338 patients were included (mean age of 63.4 years, range: 33-88). Of these, 1.0% had MBC (1826/176,338) and 99.0% were female patients (174,512/176,338). MBC patients were more likely to have increased tumour stage, nodal involvement, and grade 3 disease (all P < 0.001) In MBC patients, the mean score was 18.8 (range: 11-26) vs. 13.4 (range 0-33) in female patients (P < 0.001). In MBC patients, 22.4% had scores >30 (408/1822) versus 18.3% in female patients (31,852/174,500). In female patients, 52.0% had scores <18 (90,787/174,500) versus 47.8% in MBC (471/1822). Overall, patients with female patients were as likely to have scores <18 (OR: 1.04, 95% CI: 0.94-1.16), scores 18-30 (OR: 1.12, 95% CI: 1.00-1.26) and scores >30 (OR: 0.69, 95% CI: 0.45-1.07) as MBC patients., Conclusion: There are similar anticipated scores for female and MBC undergoing 21-gene expression assay testing for early stage, ER+/HER2-breast cancer. In the absence of stage matching, cautious interpretation of these results is required. Validation of the 21-gene assay in MBC is still required., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Human epidermal growth factor receptor-2 (HER2) is overexpressed in 20-25% of breast cancers. Complete eradication of disease following neoadjuvant therapies and chemotherapy has been referred to as pathological complete response (pCR)., Aims: To determine clinicopathological predictors of pCR to neoadjuvant therapies and to evaluate pCR as a surrogate to enhanced survival., Methods: Consecutive female patients with HER2 positive (HER+) breast cancer managed surgically in a single institution between 2005 and 2015 were included. Descriptive statistics and binary logistic regression were used to determine predictors of pCR. Appraisal of pCR as a predictor of survival was performed using Kaplan-Meier curves and Cox regression analysis., Results: 451 patients were included with a mean age of 56.6 ± 13.4 years (range 23-95). Disease-free (DFS) and overall survival (OS) was 82.3% (371/451) and 82.6% (376/451) respectively with a median follow-up of 108.0 months (range 3-184.0). 118 were treated in the neoadjuvant setting (26.2%): tumour size <50 mm (Odds Ratio (OR): 12.156, P = 0.023) and progesterone receptor negativity (OR: 2.762, P = 0.008) independently predicted breast pCR, while ductal carcinoma (OR: 3.203, P = 0.030) and grade 3 disease (OR: 2.788, P = 0.018) predicted axillary pCR. Both breast and axillary pCR predicted enhanced DFS (Hazard Ratio (HR): 0.470 & HR: 0.449) and OS (HR: 0.383 & HR: 0.307). Axillary pCR independently predicted improved OS (HR: 0.326)., Conclusion: pCR is sensitive biomarker and surrogate to survival outcomes in HER2+ breast cancer. Patients likely to achieve pCR may be predicted from traditional clinicopathological characteristics and molecular parameters., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody testing in community settings may help us better understand the immune response to this virus and, therefore, help guide public health efforts., Aim: To conduct a seroprevalence study of immunoglobulin G (IgG) antibodies in Irish GP clinics., Design & Setting: Participants were 172 staff and 799 patients from 15 general practices in the Midwest region of Ireland., Method: This seroprevalence study utilised two manufacturers' point-of-care (POC) SARS-CoV-2 immunoglobulin M (IgM)-IgG combined antibody tests, which were offered to patients and staff in general practice from 15 June to 10 July 2020., Results: IgG seroprevalence was 12.6% in patients attending general practice and 11.1% in staff working in general practice, with administrative staff having the lowest seroprevalence at 2.5% and nursing staff having the highest at 17.6%. Previous symptoms suggestive of COVID-19 and history of a polymerase chain reaction (PCR) test were associated with higher seroprevalence. IgG antibodies were detected in approximately 80% of participants who had a previous PCR-confirmed infection. Average length of time between participants' positive PCR test and positive IgG antibody test was 83 days., Conclusion: Patients and healthcare staff in general practice in Ireland had relatively high rates of IgG to SARS-CoV-2 compared with the national average between 15 June and 10 July 2020 (1.7%). Four-fifths of participants with a history of confirmed COVID-19 disease still had detectable antibodies an average of 12 weeks post-infection. While not proof of immunity, SARS-CoV-2 POC testing can be used to estimate IgG seroprevalence in general practice settings., (Copyright © 2021, The Authors.)

The age-related decline in functional capability is preceded by a reduction in muscle quality. The purpose of this study was to assess the combined effects of progressive resistance training (PRT) and protein supplementation beyond habitual intakes on upper leg lean tissue mass (LTM), muscle quality and functional capability in healthy 50-70 years women. In a single-blinded, randomized, controlled design, 57 healthy older women (age 61.1 ± 5.1 years, 1.61 ± 0.65 m, 65.3 ± 15.3 kg) consumed 0.33 g/kg body mass of a milk-based protein matrix (PRO) for 12 weeks. Of the 57 women, 29 also engaged in a PRT intervention (PRO + PRT). In comparison to the PRO group (n = 28), those in the PRO + PRT group had an increase in upper leg LTM [0.04 (95% CI -0.07 to 0.01) kg vs. 0.13 (95% CI 0.08-0.18) kg, P = 0.027], as measured by Dual-energy X-ray absorptiometry; an increase in knee extensor (KE) torque [-1.6 (95% CI -7.3 to 4.4 N m) vs. 10.2 (95% CI 4.3-15.8 N m), P = 0.007], as measured from a maximal voluntary isometric contraction (Con-Trex MJ; CMV AG); and an increase in extended gait speed [-0.01 (95% CI -0.52-0.04) m s -1 vs. 0.10 (95% CI 0.05-0.22) m s -1 , P = 0.001] as measured from a maximal 900 m effort. There was no difference between groups in the time taken to complete 5 chair rises or the number of chair rises performed in 30 s (P > 0.05). PRT in healthy older women ingesting a dietary protein supplement is an effective strategy to improve upper leg LTM, KE torque and extended gait speed in healthy older women.

Background: Key areas of research on the preservation of lean tissue mass (LTM) during aging are determinations of the protein requirement and optimal protein intake at meals., Objective: The aim of this study was to determine the effect of protein supplementation at breakfast and lunch for 24 wk beyond habitual intakes on whole-body LTM in healthy adults aged 50-70 y., Methods: In a single-blinded, randomized, controlled design, 60 healthy older men and women (aged 61 ± 5 y) with a body mass index (in kg/m(2)) of 25.8 ± 3.6 consumed either 0.165 g/kg body mass of a milk-based protein matrix (PRO) or an isoenergetic, nonnitrogenous maltodextrin control (CON) at breakfast and midday meals, the lower protein-containing meals of the day, for 24 wk. Dual-energy X-ray absorptiometry was used to measure the change in LTM., Results: After the intervention, protein intake in the PRO group increased from 0.23 ± 0.1 to 0.40 ± 0.1 g/kg for breakfast and from 0.31 ± 0.2 to 0.47 ± 2 g/kg for the midday meal. In response, LTM increased by 0.45 (95% CI: 0.06, 0.83) kg in the PRO group compared with a decrease of 0.16 (95% CI: -0.49, 0.17) kg in the CON group (P = 0.006). Appendicular LTM accounted for the majority of the difference in LTM, increasing by 0.27 (95% CI: 0.05, 0.48) kg in the PRO group compared with no change in the CON group (P = 0.002)., Conclusions: Protein supplementation at breakfast and lunch for 24 wk in healthy older adults resulted in a positive (+0.6 kg) difference in LTM compared with an isoenergetic, nonnitrogenous maltodextrin control. These observations suggest that an optimized and balanced distribution of meal protein intakes could be beneficial in the preservation of lean tissue mass in the elderly. This trial was registered at clinicaltrials.gov as NCT02529124., (© 2016 American Society for Nutrition.)

Gas chromatography with electron capture detection (GC-ECD) analysis of explosive-related nitro organic compounds was performed using four different column stationary phases with the focus being on their impact on analyte stability and transfer efficiency during analysis. All four columns used were 6 m x 0.53 mm, and the four stationary phases were a 1.0-microm thick 5% phenyl siloxane/95% methyl siloxane non-polar phase, a 1.5-microm thick 5% phenyl siloxane/95% methyl siloxane non-polar phase optimized for explosives analysis, an intermediate polarity 0.5-microm thick trifluoropropylmethyl siloxane phase, and a proprietary intermediate polarity 0.5-microm thick phase. Although all exhibited similar recovery (as defined as the detector signal per injected mass) when new, the intermediate polarity phases maintained higher sample recovery over the course of analyzing hundreds of samples than the non-polar phases, particularly for the nitramines hexahydro-1,3,5-trinitro-1,3,5-triazine and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine, for which a 7x and 3x decrease in recovery were observed, and the nitrate esters nitroglycerin and pentaerythritol tetranitrate, for which a 7x and 11x decrease in recovery were observed. For most other explosive-related compounds, the differences in recovery were between 1.5x and 3x over the same course. Although the detailed chemical formulation of the stationary phases have not been disclosed by their manufacturers, we attribute the observed differences in performance to the stability of their passivation chemistries with regard to other mobile-phase compounds contained in complex field samples. Although these effects have been qualitatively noted in the past and in response, maintenance procedures have been developed to help account for this behavior, the analyst's preference is to use an explosives analysis method that does not require these time-consuming measures. Our desire to prolong this maintenance interval provided the motivation for the assessment reported in this paper. From our assessment, we conclude that manufacturers of GC columns should focus more attention on the stationary phase and passivation chemistries that can lead to the development of a column that is better able to maintain passivation against explosive compound degradation; and users intending to perform large numbers of analyses using GC-ECD should make this a consideration when selecting a column.