HIV-1 genotypic resistance testing using the Vela automated next-generation sequencing platform

HIV-1 genotypic resistance testing using the Vela automated next-generation sequencing platform

The feasibility of producing mitochondrial DNA (mtDNA) knowledge has expanded significantly with the introduction of next-generation sequencing (NGS), particularly in the technology of whole mtDNA genome (mitogenome) sequences. However, the evaluation of those knowledge has emerged as the best problem to implementation in forensics.

To deal with this want, a customized toolkit to be used in the CLC Genomics Workbench (QIAGEN, Hilden, Germany) was developed by means of a collaborative effort between the Armed Forces Medical Examiner System – Armed Forces DNA Identification Laboratory (AFMES-AFDIL) and QIAGEN Bioinformatics.

Versatile ion S5XL sequencer for focused subsequent technology sequencing of strong tumors in a scientific laboratory

The AFDIL-QIAGEN mtDNA Expert, or AQME, generates an editable mtDNA profile that employs forensic conventions and consists of the interpretation vary required for mtDNA knowledge reporting. AQME additionally integrates an mtDNA haplogroup estimate into the evaluation workflow, which gives the analyst with phylogenetic nomenclature steerage and a profile high quality verify with out the use of an exterior software.

HIV-1 genotypic resistance testing using the Vela automated next-generation sequencing platform
HIV-1 genotypic resistance testing using the Vela automated next-generation sequencing platform

Supplemental AQME outputs reminiscent of nucleotide-per-position metrics, configurable export information, and an audit path are produced to help the analyst throughout overview. AQME is utilized to straightforward CLC outputs and thus might be integrated into any mtDNA bioinformatics pipeline inside CLC no matter pattern sort, librarypreparation or NGS platform.

An analysis of AQME was carried out to exhibit its performance and reliability for the evaluation of mitogenome NGS knowledge. The research analyzed Illumina mitogenome knowledge from 21 samples (together with related controls) of various high quality and pattern preparations with the AQME toolkit.

A complete of 211 software edits had been routinely utilized to 130 of the 698 complete variants reported in an effort to stick to forensic nomenclature. Although further guide edits had been required for 3 samples, supplemental instruments reminiscent of mtDNA haplogroup estimation assisted in figuring out and guiding these essential modifications to the AQME-generated profile.

Along with profile technology, AQME reported correct haplogroups for 18 of the 19 samples analyzed. The single errant haplogroup project, though phylogenetically shut, recognized a bug that solely impacts partial mitogenome knowledge. Future changes to AQME’s haplogrouping software will deal with this bug in addition to improve the general scoring technique to raised refine and automate haplogroup assignments.

As NGS permits broader use of the mtDNA locus in forensics, the availability of AQME and different forensic-focused mtDNA evaluation instruments will ease the transition and additional help mitogenome evaluation inside routine casework. Toward this finish, the AFMES-AFDIL has utilized the AQME toolbox along with the CLC Genomics Workbench to efficiently validate and implement two NGS mitogenome strategies.


Next technology sequencing primarily based tumor tissue genotyping entails complicated workflow and a comparatively longer turnaround time. Semiconductor primarily based subsequent technology platforms assorted from low throughput Ion PGM to excessive throughput Ion Proton and Ion S5XL sequencer. In this research, we in contrast Ion PGM and Ion Proton, with a brand new Ion S5XL NGSsystem for workflow scalability, analytical sensitivity and specificity, turnaround time and sequencing efficiency in a scientific laboratory.


Eighteen strong tumor samples constructive for numerous mutations as detected beforehand by Ion PGM and Ion Proton had been chosen for research. Libraries had been ready using DNA (vary10-40ng) from micro-dissected formalin-fixed, paraffin-embedded (FFPE) specimens using the Ion Ampliseq Library Kit for complete most cancers (CCP), oncomine complete most cancers (OCP) and most cancers hotspot panel v2 (CHPv2) panel as per producer’s directions.

The CHPv2 had been sequenced using Ion PGM whereas CCP and OCP had been sequenced using Ion Proton respectively. All the three libraries had been additional sequenced individually (S540) or multiplexed (S530) using Ion S5XL. For S5XL, Ion chef was used to automate template preparation, enrichment of ion spheres and chip loading. Data evaluation was carried out using Torrent Suite 4.6 software program on board S5XL and Ion Reporter.

A restrict of detection and reproducibility research was carried out using serially diluted DLD1 cell line.RESULTSA complete of 241 variant calls (235 single nucleotide variants and 6 indels) anticipated in the studied cohort had been efficiently detected by S5XL with 100% and 97% concordance with Ion PGM and Proton, respectively. Sequencing run time was decreased from 4.5 to 2.5 hours with output vary of 3-5 GB (S530) and 8-9.3Gb (S540). Data evaluation time for the Ion S5XL is quicker 1 h (S520), 2.5 h (S530) and 5 h (S540) chip, respectively as in comparison with the Ion PGM (3.5-5 h) and Ion Proton (8h). A restrict detection of 5% allelic frequency was established together with excessive inter-run reproducibility.


SIon S5XL system simplified workflow in a scientific laboratory, was possible for operating smaller and bigger panels on the similar instrument, had a shorter turnaround time, and confirmed good concordance for variant calls with comparable sensitivity and reproducibility as the Ion PGM and Proton.

High throughput sequencing of T-cell receptor repertoire using dry blood spots

High throughput sequencing of T-cell receptor repertoire using dry blood spots

Immunology analysis, significantly subsequent technology sequencing (NGS) of the immune T-cell receptor β (TCRβ) repertoire, has superior development in a number of fields, together with therapy of varied cancers and autoimmune ailments. This research aimed to establish the TCR repertoires from dry blood spots (DBS), a technique that can assist gathering real-world knowledge for biomarker functions.Finger-prick blood was collected onto a Whatman filter card.

RNA was extracted from DBS of the filter card, and absolutely automated multiplex PCR was carried out to generate a TCRβ chain library for subsequent technology sequencing (NGS) evaluation of distinctive CDR3s (uCDR3).We demonstrated that the dominant clonotypes from the DBS outcomes recapitulated these present in complete blood.

According to the statistical evaluation and laboratory affirmation, 40 of 2-mm punch disks from the filter playing cards had been sufficient to detect the shared prime clones and have robust correlation within the uCDR3 discovery with complete blood. uCDR3 discovery was neither affected by storage temperatures (room temperature versus – 20 °C) nor storage durations (1, 14, and 28 days) when in comparison with complete blood.

High throughput sequencing of T-cell receptor repertoire using dry blood spots
High throughput sequencing of T-cell receptor repertoire using dry blood spots

About 74-90% of prime 50 uCDR3 clones of complete blood is also detected from DBS. A low price of clonotype sharing, 0.03-1.5%, was discovered amongst completely different people.The DBS-based TCR repertoire profiling methodology is minimally invasive, supplies handy sampling, and incorporates absolutely automatedlibrarypreparation.

The system is delicate to low RNA enter, and the outcomes are extremely correlated with complete blood uCDR3 discovery permitting research scale-up to higher perceive the connection and mutual influences between the immune and ailments.

Fully automated pattern preparation process to measure medication of abuse in plasma by liquid chromatography tandem mass spectrometry

For the evaluation of medication and pharmaceutical compounds in organic matrices, extraction procedures are usually used for LC-MS/MS evaluation usually requiring handbook steps in pattern preparation. In this research, we report a completely automated extraction methodology carried out by a programable liquid handler straight coupled to an LC-MS/MS system for the willpower of 42 elements (illicit medication and/or metabolites) (plus 20 deuterated inner requirements).

The acquisition was carried out in optimistic ionization mode with as much as 15 MRM transitions per compound, every with optimized collision power (MRM spectrum mode) to allow qualitative library searching along with quantitation.

After placing the pattern tube into the systemno additional intervention was obligatory: automatedpreparation used 50 μL of blood or plasma with three μL of extracted pattern injected for evaluation. The methodology was validated according to the necessities of ISO 15189.

The restrict of detection and quantification was 1-5 ng/mL depending on the compound. Stability experiments discovered that historic calibration curve knowledge information may precisely quantify for as much as 1 month with lower than 20% uncertainty.

Comparison to a QuEChERS methodology was made using affected person samples providing a regression correlation R2 = 0.98 between the 2 strategies. This method was efficiently designed to assist parallel pattern preparation and evaluation subsequently considerably increasing pattern throughput and diminished cycle instances. Graphical summary ᅟ.

Sample Quality Control of Cell-Free DNA

Sample Quality Control of Cell-Free DNA

Quality management of nucleic acid beginning materials is crucial to make sure the success of downstream experiments. Especially, Next Generation Sequencing (NGS) developed to a robust device in virtually all genetic analysis and diagnostic areas. Due to the institution of low enter library protocols for NGS workflows sequencing of cell-free DNA (cfDNA) grew to become doable.

Since the downstream purposes are sometimes time-consuming and costly, tight QC steps are required to make sure that samples are match for functions. These QC steps may be carried out with automated electrophoresis programs. Different cell-free DNA samples had been evaluated for Sample high quality with an Agilent 4200 TapeStation system and the Agilent Cell-free DNA ScreenTape assay. Depending on preanalytical pattern therapy or extraction strategies the standard of cfDNA can fluctuate.

The outcomes embrace a rating to qualify cfDNA samples in response to their contamination stage with excessive molecular weight materials. This permits defining a threshold for goal pattern qualification previous to librarypreparation. Moreover, correct quantification of cfDNA samples is crucial to find out appropriate enter quantities for cfDNA librarypreparation previous to sequencing.

 Sample Quality Control of Cell-Free DNA
Sample Quality Control of Cell-Free DNA

Quality management of cfDNA is crucial to make sure the success of downstream experiments. Automated electrophoresis programs standardize pattern high quality management and allow goal pattern integrity evaluation in addition to the institution of high quality thresholds.

Automated Workflow for Somatic and Germline Next Generation Sequencing Analysis in Routine Clinical Cancer Diagnostics

Thanks to customized drugs developments and collaborations between trade, scientific analysis teams and regulatory companies, subsequent technology sequencing (NGS) is popping into a standard observe quicker than one might have initially anticipated.

When contemplating scientific purposes of NGS in oncology, a fast workflow for DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue samples, in addition to producing prime quality librarypreparation, may be actual challenges. Here we take into account these targets and the way making use of efficient automation expertise to NGS workflows could assist enhance yield, timing and quality-control. We firstly evaluated DNA restoration from archived FFPE blocks from three totally different guide extraction strategies and two automated extraction workstations.

The workflow was then applied to somatic (lung/colon panel) and germline (BRCA1/2librarypreparation for NGS evaluation exploiting two automated workstations. All industrial kits gave good ends in phrases of DNA yield and high quality.

On the opposite hand, the automated workstation workflow has been confirmed to be a sound computerized extraction system to acquire prime quality DNA appropriate for NGS evaluation (lung/colon Ampli-seq panel).

Moreover, it may be effectively built-in with an open liquid dealing with platform to supply high-quality libraries from germline DNA with extra reproducibility and excessive protection for focused sequences in much less time (BRCA1/2). The introduction of automation in routine workflow results in an enchancment of NGS standardization and elevated scale up of pattern preparations, lowering labor and timing, with optimization of reagents and administration.

1 2 3