A DNA typing panel of 201 genetic markers for degraded samples: development and validation.

With the increasing number of complex forensic cases in recent years, it's more important to combine the different types of genetic markers such as short tandem repeats (STRs), single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (InDels), and microhaplotypes (MHs) to provide more genetic information. In this study, we selected totally 201 genetic markers, including 24 autosomes STRs (A-STRs), 24 Y chromosome STRs (Y-STRs), 110 A-SNPs, 24 Y-SNPs, 9 A-InDels, 1 Y-InDel, 8 MHs, and Amelogenin to establish the HID_AM Panel v1.0, a Next-Generation Sequencing (NGS) detection system. According to the validation guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), the repeatability, accuracy, sensitivity, suitability for degraded samples, species specificity, and inhibitor resistance of this system were assessed. The typing results on 48 STRs and Amelogenin of this system were completely consistent with those obtained using capillary electrophoresis. This system accurately detected 79 SNPs as parallelly confirmed by a FGx sequencer with the ForenSeq™ DNA Signature Prep Kit. Complete allele typing results could be obtained with a DNA input of no less than 200 pg. The detection success rate of this system was significantly higher than that of the GlobalFiler™ kit when the degradation index of mock degraded sample was greater than 15.87. When the concentration of hematin in the amplification system was ≤40 µmol/L, indigo blue was ≤2 mmol/L, or humic acid was ≤15 ng/µL, amplification was not significantly inhibited. The system barely amplified the DNA extract from duck, mouse, cow, rabbit, and chick. The detection rate of STRs on routine samples of this panel is 99.74%, while all the SNPs, InDels, and MHs were successfully detected. In summary, we setup a NGS individual typing panel including 201 genetic markers with the high accuracy, sensitivity, species specificity, and inhibitors resistance, which is applicable for individual identification of degraded samples.

Rapid genotyping of 32 insertion/deletion panel for human identification using fluorogenic probes-based multiplex real-time PCR.

BACKGROUND: Insertion and deletion (InDel) polymorphisms have considerable potential in the field of forensic genetics because of their low mutation rate and small amplicons. At present, InDel polymorphisms detection based on the technique of capillary electrophoresis is the main technique used in forensic DNA laboratory. However, this method is complicated and time-consuming, and is not suitable for rapid on-site paternity and personal identification. Next-generation sequencing analysis of InDels polymorphisms requires expensive instruments, large upfront reagent and supply costs, computational requirements and complex bioinformatics, increased the time to obtain results. Thus, there is an urgent need to establish a method to provide reliable, rapid, sensitive and economical genotyping for InDels. METHOD: A rapid InDels (32 InDels) panel was established using fluorogenic probes-based multiplex real-time PCR with microfluidic test cartridge and portable real-time PCR instrument. Then, we performed several validation studies including concordance, accuracy, sensitivity, stability, species specificity. RESULTS: It showed that the complete genotypes could be obtained from ≥100 pg of input DNA and from a series of challenging samples with high accuracy and specificity within 90 min. CONCLUSION: This method provides a rapid and cost-effective solution for InDels genotyping and personal identification in portable format.

Genetic polymorphisms of 66 InDel loci in the Chinese Han population.

Insertion/deletion polymorphism (InDel) genetic markers refer to insertion or deletion of DNA fragments into genomic DNA, which have advantages in the identification of degraded samples. In this study, we independently screened 66 highly polymorphic InDel markers from the dbSNP database to establish a multiplex PCR system for forensic DNA identification using next-generation sequencing system (66-plex InDels). We assessed the population genetic data among 251 Chinese Han population using this system and evaluated their potential forensic application. The results showed that all 66 InDel loci conformed to the Hardy-Weinberg equilibrium (P>0.000 758), and all the pairwise InDel loci were in linkage equilibrium after Bonferroni correction. The mean observed heterozygosity (Ho) was 0.482, the mean expected heterozygosity (He) was 0.483,the mean discrimination power (DP) was 0.612, the mean polymorphism information content (PIC) was 0.365, the total discrimination power (TDP) reached 0.999 999 999 999 999 999 999 999 999 428 18. The cumulative power of exclusion for 66 InDel loci was 0.999 739 in duo cases (CPEduo) and was 0.999 999 999 417 in trios cases (CPEtrio). The results show that the 66 InDel loci have high genetic polymorphisms in the Chinese Han population and can be used independently for forensic DNA identification and paternity testing.

[Diagnosis and treatment for postoperative lobar torsion].

OBJECTIVE: To analyze retrospectively 8 cases of postoperative lobar torsion after thoracotomy. METHODS: 8 cases of postoperative lobar torsion were collected (5 men and 3 women; median age, 55.0 +/- 7.7 years), including lobectomy 4 (left upper lobe of lung 2, right upper lobe of lung 2), esophageal carcinosectomy 2, resection of schwannoma in the right upper mediastinum 1, and descending aorta replacement 1. RESULTS: The postoperative lobar torsions were right middle lobe 2, right upper lobe 1, left upper lobe 3, left lower lobe 1, left lung 1. The median peak temperature was 38.4 degrees C (range, 37.8 - 40.2 degrees C) and the median white blood cell count was 10.6 x 10(9) cells/L (range, 9.3 - 14.9 x 10(9) cell/L) during the first 48 hours postoperatively. Postoperative radiographs demonstrated pulmonary infiltrates and volume loss in 6 patients and complete opacification in 2 patients. The diagnosis of lobar torsion was made a median of 4 days (range, 2 - 14 days) after the initial operation; 6 patients underwent resection of lung and recovered; 2 had the injured lobe or lung rotated and died. Complications after reoperation included respiratory failure in 2 patients, atrial arrhythmia in 2 patients. Median hospitalization was 24 days and range from 10 to 56 days. CONCLUSIONS: The mobilization of hilus of lung or residual pulmonary atelectasis is the main mechanism of the lobar torsion after thoracotomy. Lobar torsion represents a difficult diagnostic dilemma in the early postoperative period after thoracotomy. Exploratory thoracotomy must be performed without delay. The injured parenchyma should be sacrificed unless the diagnosis is obtained very early. When the injured lobe or lung is rotated back into normal position, simultaneous endotracheal suction is very important to prevent aspiration of fluid from the obstructed part of the bronchial tree to the uninvolved segments and dangerous postoperative hypoxia.