Cost-Effective Next Generation Sequencing-Based STR Typing with Improved Analysis of Minor, Degraded and Inhibitor-Containing DNA Samples.

Forensic DNA profiles are established by multiplex PCR amplification of a set of highly variable short tandem repeat (STR) loci followed by capillary electrophoresis (CE) as a means to assign alleles to PCR products of differential length. Recently, CE analysis of STR amplicons has been supplemented by high-throughput next generation sequencing (NGS) techniques that are able to detect isoalleles bearing sequence polymorphisms and allow for an improved analysis of degraded DNA. Several such assays have been commercialised and validated for forensic applications. However, these systems are cost-effective only when applied to high numbers of samples. We report here an alternative, cost-efficient shallow-sequence output NGS assay called maSTR assay that, in conjunction with a dedicated bioinformatics pipeline called SNiPSTR, can be implemented with standard NGS instrumentation. In a back-to-back comparison with a CE-based, commercial forensic STR kit, we find that for samples with low DNA content, with mixed DNA from different individuals, or containing PCR inhibitors, the maSTR assay performs equally well, and with degraded DNA is superior to CE-based analysis. Thus, the maSTR assay is a simple, robust and cost-efficient NGS-based STR typing method applicable for human identification in forensic and biomedical contexts.

Video clip assessment of a salivary gland ultrasound scoring system in Sjögren's syndrome using consensual definitions: an OMERACT ultrasound working group reliability exercise.

OBJECTIVE: To develop ultrasound (US) definitions and a US novel scoring system for major salivary gland (SG) lesions in patients with primary Sjögren's syndrome (pSS) and to test their intrareader and inter-reader reliability using US video clips. METHODS: Twenty-five rheumatologists were subjected to a three-round, web-based Delphi process in order to agree on (1) definitions and scanning procedure of salivary gland ultrasonography (SGUS): parotid, submandibular and sublingual glands (PG, SMG and SLG); (2) definitions for the elementary SGUS lesions in patients with Sjögren's syndrome; (3) scoring system for grading changes. The experts rated the statements on a 1-5 Likert scale. In the second step, SGUS video clips of patients with pSS and non-pSS sicca cases were collected containing various spectrums of disease severity followed by an intrareader and inter-reader reliability exercise. Each video clip was evaluated according to the agreed definitions. RESULTS: Consensual definitions were developed after three Delphi rounds. Among the three selected SGs, US assessment of PGs and SMGs was agreed on. Agreement was reached to score only greyscale lesions and to focus on anechoic/hypoechoic foci in a semiquantitative matter or, if not possible on a qualitatively (present/absent) evaluation of fatty or fibrous lesions. Intrareader reliability for detecting and scoring these lesions was excellent (Cohen's kappa 0.81) and inter-reader reliability was good (Light's kappa 0.66). CONCLUSION: New definitions for developing a novel semiquantitative US score in patients with pSS were developed and tested on video clips. Inter-reader and intrareader reliabilities were good and excellent, respectively.

Toward Quantitative Assessment of Rheumatoid Arthritis Using Volumetric Ultrasound.

GOAL: Rheumatoid arthritis (RA) is characterized by inflammation within the joint space as well as erosion or destruction of the bone surface. We believe that volumetric (3-D) ultrasound imaging of the joints in conjunction with automated image-analysis tools for segmenting and quantifying the regions of interest can lead to improved RA assessment. METHODS: In this paper, we describe our proposed algorithms for segmenting 1) the 3 -D bone surface and 2) the 3-D joint capsule region. We improve and extend previous 2-D bone extraction methods to 3-D and make our algorithm more robust to the intensity loss due to surface normals facing away from incident acoustic beams. The extracted bone surfaces coupled with a joint-specific anatomical model are used to initialize a coarse localization of the joint capsule region. The joint capsule segmentation is refined iteratively utilizing a probabilistic speckle model. RESULTS: We apply our methods on 51 volumes from 8 subjects, and validate segmentation results with expert annotations. We also provide the quantitative comparison of our bone detection with magnetic resonance imaging. These automated methods have achieved average sensitivity/precision rates of 94%/93% for bone surface detection, and 87%/83% for joint capsule segmentation. Segmentations of normal and inflamed joints are compared to demonstrate the potential of using proposed tools to assess RA pathology at the joint level. CONCLUSION: The proposed image-analysis methods showed encouraging results as compared to expert annotations. SIGNIFICANCE: These computer-assisted tools can be used to help visualize 3-D anatomy in joints and help develop quantitative measurements toward RA assessment.

Musculoskeletal ultrasound training and competency assessment program for rheumatology fellows.

OBJECTIVES: The purpose of this study was to establish standards for musculoskeletal ultrasound competency through knowledge and skills testing using criterion-referenced methods. METHODS: Two groups of rheumatology fellows trained in musculoskeletal ultrasound through a standardized curriculum, which required submission of ultrasound studies for review over 8 months. Both groups then completed written and practical examinations in musculoskeletal ultrasound. Instructors, advanced users, and intermediate users of musculoskeletal ultrasound served as comparison groups. A passing score (competency) was established for the written examination by the Angoff procedure and for the practical examination by the borderline method. RESULTS: Thirty-eight fellows (19 in each group) took the final examination. Five fellows failed the written examination, and 1 failed the practical examination, whereas none of the advanced users failed. Written examination scores did not differ between the two fellow groups (74% versus 70%; P > .05), were reliable, and were able to discriminate between the intermediate and advanced groups. Practical and written examination results correlated in both groups (first group, r = 0.70; P = .0008; second group, r = 0.59; P = .009). CONCLUSIONS: Criterion-referenced methods were used for the first time to determine fellow musculoskeletal ultrasound competency. The examination used to determine competency was reproducible, was reliable, and could differentiate musculoskeletal ultrasound users with different levels of experience. Most rheumatology fellows completing our program passed the written and practical examinations, suggesting achievement of basic musculoskeletal ultrasound competency.