An accessible, efficient and global approach for the large-scale sequencing of bacterial genomes

We have developed an efficient and inexpensive pipeline for streamlining large-scale collection and genome sequencing of bacterial isolates. Evaluation of this method involved a worldwide research collaboration focused on the model organism Salmonella enterica, the 10KSG consortium. Following the optimization of a logistics pipeline that involved shipping isolates as thermolysates in ambient conditions, the project assembled a diverse collection of 10,419 isolates from low- and middle-income countries. The genomes were sequenced using the LITE pipeline for library construction, with a total reagent cost of less than USD$10 per genome. Our method can be applied to other large bacterial collections to underpin global collaborations.

Comparison of Initial pH Adjustment Prior to Thermophilic Anaerobic Digestion of Lime-Treated Corn Stover via Liquid Digestate or CO2

Neutralization with liquid digestate and CO2 was compared herein to adjust the pH of lime-treated corn stover. The effects on the thermophilic (55 °C) anaerobic digestion were also analyzed. Liquid digestate neutralization (LDN) caused a decrease in pH from 10.5 to 7.5 in 60 h and accumulation of acetic/isobutyric acids. The CO2 neutralization (CN) under solid-state conditions reduced the pH from 10.5 to 8.5 in 30 min, which is faster than that of LDN and did not affect the subsequent anaerobic digestion. Biomethane production rate indicates that LDN contributed to the performance of anaerobic digestion, but this was not sufficient to compensate for the loss of total biomethane yield, resulting in a negative net profit (i.e., revenue from increased energy production minus reagent cost). For CN under solid-state conditions, the biomethane production was highest in both liquid- and solid-state anaerobic digestion, and also obtained a net profit of 98.74–100.89 RMB/tonne dry biomass. Therefore, the solid-state condition CN is a more efficient and economic method for adjusting initial pH of lime-treated corn stover.

Efficiency of pooled surveillance testing in academic labs to detect and inhibit COVID-19 outbreaks

Robust surveillance testing is a key strategic plan to prevent COVID-19 outbreaks and slow the spread of the SARS-CoV-2 pandemic; however, limited resources, facilities and time often impair the implementation of a widespread surveillance effort. To mitigate these resource limitations, we employed a strategy of pooling samples, reducing reagent cost and processing time. Through utilizing academic faculty and labs, successful pooled surveillance testing was conducted throughout Fall 2020 semester to detect positive SARS-CoV-2 infections in a population of 4400 students. During the semester, over 25,000 individual COVID status evaluations were made by pooling eight individual samples into one quantitative reverse transcription polymerase chain reaction. This pooled surveillance strategy was highly effective at detecting infection and significantly reduced financial burden and cost by $3.6 million.

One-Seq: A Highly Scalable Sequencing-Based Diagnostic for SARS-CoV-2 and Other Single-Stranded Viruses

The management of pandemics such as COVID-19 requires highly scalable and sensitive viral diagnostics, together with variant identification. Next-generation sequencing (NGS) has many attractive features for highly multiplexed testing, however current sequencing-based methods are limited in throughput by early processing steps on individual samples (e.g. RNA extraction and PCR amplification). Here we report a new method, "One-Seq", that eliminates the current bottlenecks in scalability by enabling early pooling of samples, before any extraction or amplification steps. To enable early pooling, we developed a one-pot reaction for efficient reverse transcription (RT) and upfront barcoding in extraction-free clinical samples, and a "protector" strategy in which carefully designed competing oligonucleotides prevent barcode crosstalk and preserve detection of the high dynamic range of viral load in clinical samples. This method is highly sensitive, achieving a limit of detection (LoD) down to 2.5 genome copy equivalent (gce) in contrived RT samples, 10 gce in multiplexed sequencing, and 2-5 gce with multi-primer detection, suggesting an LoD of 200-500 gce/ml for clinical testing. In clinical specimens, One-Seq showed quantitative viral detection against clinical Ct values with 6 logs of linear dynamic range and detection of SARS-CoV-2 positive samples down to ~360 gce/ml. In addition, One-Seq reports a number of hotspot viral mutations at equal scalability at no extra cost. Scaling up One-Seq would allow a throughput of 100,000-1,000,000 tests per day per single clinical lab, at an estimated amortized reagent cost of $1.5 per test and turn-around time of 7.5-15 hr.

Rapid and economical drug resistance profiling with Nanopore MinION for clinical specimens with low bacillary burden of Mycobacterium tuberculosis

Objective We designed and tested a Nanopore sequencing panel for direct tuberculosis drug resistance profiling. The panel targeted 10 resistance-associated loci. We assessed the feasibility of amplifying and sequencing these loci from 23 clinical specimens with low bacillary burden. Results At least 8 loci were successfully amplified from the majority for predicting first- and second-line drug resistance (14/23, 60.87%), and the 12 specimens yielding all 10 targets were sequenced with Nanopore MinION and Illumina MiSeq. MinION sequencing data was corrected by Nanopolish and recurrent variants were filtered. A total of 67,082 bases across all consensus sequences were analyzed, with 67,019 bases called by both MinION and MiSeq as wildtype. For the 41 single nucleotide variants (SNVs) called by MiSeq with 100% variant allelic frequency (VAF), 39 (95.1%) were called by MinION. For the 22 mixed bases called by MiSeq, a SNV with the highest VAF (70%) was called by MinION. With short assay time, reasonable reagent cost as well as continuously improving sequencing chemistry and signal correction pipelines, this Nanopore method can be a viable option for direct tuberculosis drug resistance profiling in the near future.

Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore

Genome sequencing has been widely deployed to study the evolution of SARS-CoV-2 with more than 90,000 genome sequences uploaded to the GISAID database. We published a method for SARS-CoV-2 genome sequencing (https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w) online on January 22, 2020. This approach has rapidly become the most popular method for sequencing SARS-CoV-2 due to its simplicity and cost-effectiveness. Here we present improvements to the original protocol: i) an updated primer scheme with 22 additional primers to improve genome coverage, ii) a streamlined library preparation workflow which improves demultiplexing rate for up to 96 samples and reduces hands-on time by several hours and iii) cost savings which bring the reagent cost down to £10 per sample making it practical for individual labs to sequence thousands of SARS-CoV-2 genomes to support national and international genomic epidemiology efforts.

An accessible, efficient and global approach for the large-scale sequencing of bacterial genomes

We have developed an efficient and inexpensive pipeline for streamlining large-scale collection and genome sequencing of bacterial isolates. Evaluation of this method involved a worldwide research collaboration focused on the model organism Salmonella enterica, the 10KSG consortium. By optimising a logistics pipeline that collected isolates as thermolysates, permitting shipment in ambient conditions, the project assembled a diverse collection of 10,419 clinical and environmental isolates from low- and middle-income countries in less than one year. The bacteria were obtained from 51 countries/territories dating from 1949 to 2017, with a focus on Africa and Latin-America. All isolates were collected in barcoded tubes and genome sequenced using an optimised DNA extraction method and the LITE pipeline for library construction. After Illumina sequencing, the total reagent cost was less than USD$10 per genome. Our method can be applied to genome-sequence other large bacterial collections at a relatively low cost, within a limited timeframe, to support global collaborations.

Predictive molecular pathology in the time of COVID-19

AimsIn the time of COVID-19, predictive molecular pathology laboratories must still timely select oncological patients for targeted treatments. However, the need to respect social distancing measures may delay results generated by laboratory-developed tests based on sequential steps a long hands-on time. Laboratory workflows should now be simplified.MethodsThe organisation of the University of Naples Federico II predictive pathology laboratory was assessed before (March–April 2019) and during (March–April 2020) the Italian lockdown.ResultsThe number of patients undergoing single or multiple biomarker testing was similar in 2019 (n=43) and in 2020 (n=45). Considering adequate samples for molecular testing, before the outbreak, next-generation sequencing was mostly used (35/42, 83.3%). Testing six genes had a reagent cost of €98/patient. Conversely, in 2020, almost all cases (38/41, 92.7%) were analysed by automated testing. This latter had for any single assay/gene a significant reagent cost (€95–€136) and a faster mean turnaround time (5.3 vs 7.9 working days).ConclusionIn the times of coronavirus, laboratory fully automated platforms simplify predictive molecular testing. Laboratory staff may be more safely and cost-effectively managed.

Minimized double guide RNA libraries enable scale-limited CRISPR/Cas9 screens

Genetic screens based on CRISPR/Cas technology are a powerful tool for understanding cellular phenotypes. However, the coverage and replicate requirements result in large experiment sizes, which are limiting when samples are scarce, or the protocols are expensive and laborious. Here, we present an approach to reduce the scale of genome-wide perturbation screens up to fivefold without sacrificing performance. To do so, we deliver two randomly paired gRNAs into each cell, and rely on recent advances in gRNA design, as well as availability of gRNA effect measurements, to reduce the number of gRNAs per gene. We designed a human genome-wide library that has effective size of 30,000 constructs, yet targets each gene with three gRNAs. Our minimized double guide RNA library gives similar results to a standard single gRNA one, but using substantially fewer cells. We demonstrate that genome-wide screens can be optimized in a demanding model of induced pluripotent stem cells, reducing reagent cost 70% per replicate compared to conventional approach, while retaining high performance. The screen design and the reduction in scale it provides will enable functional genomics experiments across many possible combinations of environments and genetic backgrounds, as well as in hard to obtain and culture primary cells.

Effectiveness of minimum retesting intervals in managing repetitive laboratory testing: experience from a Croatian university hospital

Introduction: Inappropriate laboratory retesting can be addressed by implementing minimum retesting intervals (MRI). The aim of our study was to assess the effectiveness of the implemented MRI protocol for inpatients. Materials and methods: Minimum retesting intervals were applied for 53 laboratory tests. The overall reduction of test requests, reduction in charges and reagent cost savings, frequency of MRI alert appearance as well as the rate of MRI acceptance and ignorance were calculated for a one-year period. Reasons for violating the MRI rule, hospital departments that contributed mostly to MRI rule violation, and the frequency of MRI violations between routine and emergency laboratory were evaluated. Results: During the one-year period, 106,780 requests violated the MRI rule, which corresponds to 14.8% of all requests received. 13,843 requests were cancelled, yielding a 1.9% reduction of requested tests. High-volume tests, namely complete blood count, C-reactive protein, alanine aminotransferase, gamma-glutamyltransferase and total bilirubin, accounted for 65% of all generated alerts and had the highest alert ignorance (>85%). The highest cancellation rate was observed for tumor markers and autoimmunity tests, for most being at least 50%. Annual charge reduction was 62,641 EUR while reagent cost savings were 11,408 EUR. Tests performed in the emergency laboratory had a higher alert appearance than the same routine tests. The most common reason for MRI violation was clinical justification based on the patient’s condition. Most frequently ignored MRI alerts were in the intensive care unit. Conclusion: MRI implementation showed limited effectiveness in reducing testing repetition and achieving financial savings, yet provided the basis for future improvements.