Final Library Research Articles

The capacity for oestrogen to influence obesity through brown adipose tissue thermogenesis in animal models: A systematic review and meta-analysis.

SummaryPharmacological interventions to aid weight loss have historically targeted either appetite suppression or increased metabolic rate. Brown adipose tissue (BAT) possesses the capacity to expend energy in a futile cycle, thus increasing basal metabolic rate. In animal models, oestrogen has been implicated in the regulation of body weight, and it is hypothesized that oestrogen is acting by modulating BAT metabolism. A systematic search was performed, to identify research articles implementing in vivo oestrogen‐related interventions and reporting outcome measures that provide direct or indirect measures of BAT metabolism. Meta‐analyses were conducted where sufficient data were available. The final library of 67 articles were predominantly in rodent models and provided mostly indirect measures of BAT metabolism. Results of this review found that oestrogen's effects on body weight, in rats and possibly mice, are likely facilitated by both metabolic and appetitive mechanisms but are largely only found in ovariectomized models. There is a need for further studies to clarify the potential effects of oestrogen on BAT metabolism in gonad‐intact and castrated male animal models.

ULTRALOW COVERAGE SEQUENCING IS THE MOST ACCURATE LIBRARY QUANTIFICATION METHOD PRIOR TO EXOME SEQUENCING

Accurate DNA library quantification is very important in post-pooling captured exome sequencing. Underrepresented libraries will need additional sequencing, which takes extra time and money, whereas overexpressed DNA libraries can lead to the generation of more data than required, which leads to the waste of sequence capacity and a reduced number of samples per batch. There is a number of methods available to quantify DNA libraries prior to sequencings, such as UV absorption, use of intercalating dyes, capillary electrophoresis, 5’-hydrolysis probes (TaqMan©) coupled with quantitative PCR (qPCR) or droplet digital PCR. But there is no gold standard for the quantification of DNA libraries. This study compares common library quantification methods, including LabChip (PerkinElmer Inc., MA, USA), Qubit 3.0 (Thermo Fisher Scientific, MA, USA), several qPCR approaches, and ultralow coverage sequencing on Illumina MiSeq platform (with and without insert size correction). DNA libraries were prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs, MA, USA). To compare the above-mentioned approaches, cost, time, and quantification accuracy were assessed in our study. Quantification methods involving the use of Qubit and MiSeq were found to be better than qPCR and LabChip approaches at predicting the final library concentration. It was also revealed that MiSeq with insert size correction was the most accurate method for library quantification prior to exome sequencing. This method allows for correction shifts in the ratio due to enrichment. Ultralow coverage sequencing on the Illumina MiSeq platform is the most accurate method of library quantification prior to pooling and post-pooling exome enrichment.

Parallel Solution Phase Synthesis and Preliminary Biological Activity of a 5'-Substituted Cytidine Analog Library.

A 109-membered library of 5'-substituted cytidine analogs was synthesized, via funding through the NIH Roadmap Initiative and the Pilot Scale Library (PSL) Program. Reaction core compounds contained -NH2 (2) and -COOH (44 and 93) groups that were coupled to a diversity of reactants in a parallel, solution phase format to produce the target library. The assorted reactants included -NH2, -CHO, -SO2Cl, and -COOH functional groups, and condensation with the intermediate core materials 2 and 44 followed by acidic hydrolysis produced 3-91 in good yields and high purity. Linkage of the amino terminus of d-phenylalanine methyl ester to the free 5'-COOH of 44 and NaOH treatment led to core library -COOH precursor 93. In a libraries from libraries approach, compound 93 served as the vital building block for our unique library of dipeptidyl cytidine analogs 94-114 through amide coupling of the -COOH group with numerous commercial amines followed by acidic deprotection. Initial screening of the complete final library through the MLPCN program revealed a modest number of hits over diverse biological processes. These hits might be considered as starting points for hit-to-lead optimization and development studies.

Accurate mass and retention time library of serum lipids for type 1 diabetes research.

Dysregulated lipid species are linked to various disease pathologies and implicated as potential biomarkers for type 1 diabetes (T1D). However, it is challenging to comprehensively profile the blood specimen lipidome with full structural details of every lipid molecule. The commonly used reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS)-based lipidomics approach is powerful for the separation of individual lipid species, but lipids belonging to different classes may still co-elute and result in ion suppression and misidentification of lipids. Using offline mixed-mode and RPLC-based two-dimensional separations coupled with MS/MS, a comprehensive lipidomic profiling was performed on human sera pooled from healthy and T1D subjects. The elution order of lipid molecular species on RPLC showed good correlations to the total number of carbons in fatty acyl chains and total number of double bonds. This observation together with fatty acyl methyl ester analysis was used to enhance the confidence of identified lipid species. The final T1D serum lipid library database contains 753 lipid molecular species with accurate mass and RPLC retention time uniquely annotated for each of the species. This comprehensive human serum lipid library can serve as a database for high-throughput RPLC-MS-based lipidomic analysis of blood samples related to T1D and other childhood diseases. Graphical abstract.

Best Practices for Illumina Library Preparation.

In this unit, we describe a set of protocols and recommendations for Illumina library preparation. We review best practices in template quantitation methods; template fragmentation methodologies; solid-phase reverse-immobilization cleanup, including buffer exchange and size selection; end repair, A-tailing, and adapter ligation; indexing strategies; considerations regarding whether to use polymerase chain reaction; final library quantification methodologies; and normalization and pooling strategies. These workflows are applicable to both high-throughput and low-throughput Illumina library preparation and should help reduce bias, increase cost effectiveness, and produce high library yields. This is an extensive update of the previous version of this unit. © 2019 by John Wiley & Sons, Inc.

SUN-031 Quantification of Pooled Libraries for Optimizing Cluster Density in Next Generation Sequencing

Background: Next-generation sequencing (NGS) consists of massive parallel processing libraries where millions of reads are generated in a single sequencing run. To ensure efficient sequencing, the amount of DNA pool libraries must be measured precisely because it influences the density of the clusters formation. There are different quantification methods aiming to obtain high density clusters with Q30>80%, avoiding the negative effects of overclustering. Our aim was to compare the density of the clusters in exome libraries quantified by two different methodologies qPCR and MiSeq Nano V2 method. Exome libraries were prepared using SureSelect Human All Exons kits (Agilent Technology) and sequenced on Illumina HiSeq2500 platform (Illumina) using the V4 high output sequencing kit. The pools were prepared in equimolar concentrations using qPCR (KAPA Library Quantification Kit), to achieve final pool library concentration of 10 nM. Three groups were defined: the original 10 nM group (n=16); a qPCR group (n=16) in which the pool concentrations were corrected by qPCR and the MiSeq Nano V2 group (n=12) where pool concentrations were corrected by clustering data of MiSeq Nano in a direct linear proportion. The statistics analysis was performed by Kruskal-Wallis followed by Dunn test. Cluster density values are reported as the absolute distance from the optimal density (1000 k/mm2 of flow cell) specified for V4 kit. Results: The 10 nM group showed a median of 191, iqr (interquartile range) = 48-273, the qPCR group the median was 80, iqr = 30-135 and the MiSeq Nano V2 group the median was 46, iqr = 13-78. Therefore, the MiSeq NanoV2 method was significantly better than the other groups at either generating closer distances from the target cluster density value (Kruskal-Wallis test, p<0.05, H=7.967, p=0.02 with Dunn’s test, mean rank difference=13.84, p=0.01). The rate of success in the generation of data without overclustering effects was also better in Miseq Nano V2 group (Kruskal-Wallis test, p<0.05, H=7.525, p=0.02, Dunn’s multiple comparison test, mean rank difference=-9.625, p=0.01. Conclusion: The MiSeq Nano V2 method is significantly more precise to quantify the pool library concentration, increasing data yield per run, reducing potential loss of data due to inadequate clustering and covering, saving time and money in clinical research laboratories.

DOPlify, Target Sequence Enrichment and Allele Drop-Out – is there a benefit?

Introduction The ability to combine whole genome amplification (WGA) with gene-specific primers in a single PCR reaction to allow both aneuploidy detection (PGT-A) and monogenic disease detection (PGT-M) is an advantage of DOPlify® and the RHS targeted sequence enrichment (TSE) protocol. This study aims to compare Allele Drop-Out (ADO) rates by comparing three different approaches; gene specific PCR only, WGA and WGA with TSE. Material and Methods Thirty 5-cells aliquots were manually sorted from a cell line containing a 2bp heterozygous BRCA1 mutation (GM14090, Coriell Institute). To allow an assessment of aneuploidy detection, thirty 5-cell aliquots from an aneuploid cell line (GM04965, 48,XXY,+21, Coriell Institute) were also used. In the TSE approach, cells were amplified using DOPlify® (RHS Ltd) with the inclusion of three BRCA1 primers. One BRCA1 primer set was designed to specifically amplify the region on chromosome 17 containing the deletion site in GM14090 and other two primer sets to amplify BRCA1 linker markers. The resulting WGA-TSE product was diluted 1/10 and used as template for a second gene specific multiplex PCR (Qiagen) containing the same BRCA1 primers. The BRCA1 PCR products were then pooled back into the WGA-TSE product before library preparation and analysed using a single library index (WGA-TSE+GS). Secondly, for PGT-M only, cells were subjected to two rounds of gene specific PCR using multiplex PCR (Qiagen) and the same BRCA1 primers (GS+GS). Lastly, cells were amplified according to standard DOPlify® protocol (RHS Ltd) with no BRCA1 primers added. The resulting WGA product was diluted 1/10 and seeded into the BRCA1 multiplex PCR reaction (Qiagen) (WGA+GS). PCR products from the WGA-TSE+GS condition were sequenced according to standard PG-Seq™ protocol (RHS Ltd). Amplicons from the GS+GS and WGA+GS conditions were diluted 1/10 before final library pooling then were sequenced with the Illumina MiSeq. Results The 2bp heterozygous BRCA1 deletion was accurately detected in 10/10 of the WGA-TSE+GS samples, 10/10 of the GS+GS samples and 7/10 of the WGA+GS samples with average allele frequencies of 41%, 47% and 53%, respectively. The average read depth across the 2bp deletion site was 120, 129 and 138, respectively. One GM04965 sample from the GS+GS condition failed to amplify the region. Correct aneuploidy results were obtained for 9/9 BRCA1 cells and 10/10 GM04965 cells processed with the WGA-TSE+GS protocol with one BRCA1 sample excluded after failing quality control (weak amplification). The GS+GS protocol does not allow for PGT-A assessment. Conclusions The DOPlify® TSE protocol is able to reliably generate both PGT-A and PGT-M results from a cell sample representing a single embryo biopsy. The PGT-A results were not compromised, and PGT-M results showed no ADO and were comparable to the gene specific PCR protocol. These results highlight the benefit of the DOPlify® TSE protocol, which is now being clinically validated.

Guidelines for the Management of Pediatric Severe Traumatic Brain Injury, Third Edition: Update of the Brain Trauma Foundation Guidelines.

Severe Traumatic Brain Injury in Infants, Children, and Adolescents in 2019: Some Overdue Progress, Many Remaining Questions, and Exciting Ongoing Work in the Field of Traumatic Brain Injury ResearchIn this Supplement to Pediatric Critical Care Medicine, we are pleased to present the Third Edition o

The Management of Diabetes in Everyday Life (MODEL) program: development of a tailored text message intervention to improve diabetes self-care activities among underserved African-American adults.

NCT02957513.

Rapid Multiplexed Reduced Representation Bisulfite Sequencing Library Prep (rRRBS).

DNA methylation is a common mechanism of epigenetic regulation involved in transcriptional modulation and genome stability. With the evolution of next-generation sequencing technologies, establishing quantitative genome-wide DNA methylation profiles is becoming routine in many laboratories. However, many of these approaches take several days to accomplish and use subjective PCR methods to amplify sequencing libraries, which can induce amplification bias. Here we propose a rapid Reduced Representation Bisulfite Sequencing (rRRBS) protocol to minimize PCR amplification bias and reduce total time of multiplexed library construction. In this modified approach, the precise quantification of the final library amplification step is accomplished and monitored by qPCR, instead of using standard PCR and gel electrophoresis, to determine the appropriate number of cycles to perform. The main advantages of this rRRBS method are: i) Reduced amount of amplification enzyme used for library prep, ii) Reduced number of PCR cycles resulting in less PCR amplification bias, and iii) Preparation of quality multiplexed rRRBS libraries in only ~2 days.

Determination of genetic aberrations and novel transcripts involved in the pathogenesis of oligodendroglioma using array comparative genomic hybridization and next generation sequencing.

The aim of the present study was to determine the genetic aberrations and novel transcripts, particularly the fusion transcripts, involved in the pathogenesis of low-grade and anaplastic oligodendroglioma. In the present study, tissue samples were obtained from patients with oligodendroglioma and additionally from archived tissue samples from the Brain Tumor Tissue Bank of the Brain Tumor Foundation of Canada. Six samples were obtained, three of which were low-grade oligodendroglioma and the other three anaplastic oligodendroglioma. DNA and RNA were extracted from each tissue sample. The resulting genomic DNA was then hybridized using the Agilent CytoSure 4×180K oligonucleotide array. Human reference DNA and samples were labeled using Cy3 cytidine 5′-triphosphate (CTP) and Cy5 CTP, respectively, while human Cot-1 DNA was used to reduce non-specific binding. Microarray-based comparative genomic hybridization data was then analyzed for genetic aberrations using the Agilent Cytosure Interpret software v3.4.2. The total RNA isolated from each sample was mixed with oligo dT magnetic beads to enrich for poly(A) mRNA. cDNAs were then synthesized and subjected to end-repair, poly(A) addition and connected using sequencing adapters using the Illumina TruSeq RNA Sample Preparation kit. The fragments were then purified and selected as templates for polymerase chain reaction amplification. The final library was constructed with fragments between 350–450 base pairs and sequenced using deep transcriptome sequencing on an Illumina HiSeq 2500 sequencer. The array comparative genomic hybridization revealed numerous amplifications and deletions on several chromosomes in all samples. However, the most interesting result was from the next generation sequencing, where one anaplastic oligodendroglioma sample was demonstrated to have five novel fusion genes that may potentially serve a critical role in tumor pathogenesis and progression.

Reselection Yielding a Smaller and More Active Silver-Specific DNAzyme.

Ag10c is a recently reported RNA-cleaving DNAzyme obtained from in vitro selection. Its cleavage activity selectively requires Ag+ ions, and thus it has been used as a sensor for Ag+ detection. However, the previous selection yielded very limited information regarding its sequence requirement, since only ∼0.1% of the population in the final library were related to Ag10c and most other sequences were inactive. In this work, we performed a reselection by randomizing the 19 important nucleotides in Ag10c in such a way that a purine has an equal chance of being A or G, whereas a pyrimidine has an equal chance of being T or C. The round 3 library of the reselection was carefully analyzed and a statistic understanding of the relative importance of each nucleotide was obtained. At the same time, a more active mutant was identified, containing two mutated nucleotides. Further analysis indicated new base pairs leading to an enzyme with smaller catalytic loops but with ∼200% activity of the original Ag10c, and also excellent selectivity for Ag+. Therefore, a more active mutant of Ag10c was obtained and further truncations were successfully performed, which might be better candidates for developing new biosensors for silver. A deeper biochemical understanding was also obtained using this reselection method.

In silico development of new acetylcholinesterase inhibitors

In this work, we made use of fragment-based drug design (FBDD) and de novo design to obtain more powerful acetylcholinesterase (AChE) inhibitors. AChE is associated with Alzheimer’s disease (AD). It was found that the cholinergic pathways in the cerebral cortex are compromised in AD and the accompanying cholinergic deficiency contributes to the cognitive deterioration of AD patients. In the FBDD approach, fragments are docked into the active site of the protein. As fragments are molecular groups with a low number of atoms, it is possible to study their interaction with localized amino acids. Once the interactions are measured, the fragments are organized by affinity and then linked together to form new molecules with a high degree of interaction with the active site. In the other approach, we used the de novo design technique starting from reference drugs used in the AD treatment. These drugs were broken into fragments (seeds). In the growing strategy, fragments were added to each seed, growing new molecules. In the linking strategy, two or more separated seeds were linked with different fragments. Both strategies combined produced a library of more than 2 million compounds. This library was filtered using absorption, distribution, metabolism, and excretion properties. The resulting library with around six thousand compounds was filtered again. In this case, structures with Tanimoto coefficients >.85 were discarded. The final library with 1500 compounds was submitted to docking studies. As a result, 10 compounds with better interaction energy than the reference drugs were obtained.

P026 A successful breakthrough in multiplexing 11 HLA loci amplification in a single tube for NGS typing

Aim There is an increasing need to type all 11 HLA loci for solid organ and HSCT Tx to achieve better donor/recipient pairing. However, the amplification step in the existing NGS workflow is cumbersome and error-prone because it requires multiple locus-specific PCR set up for each sample. Multiplexing 11 loci in a single tube greatly simplifies the workflow. Our aims are to obtain healthy balanced coverage, and streamline the error prone steps common to PCR-based NGS assays. Methods We performed long range amplification on the genomic DNA samples using 11 loci multiplex primers that cover the entire classical Class I genes, the entire DPA1 and DQA1 genes, exon 2 to the 3’UTR of Class II genes DPB1 and DQB1, and exon 2 to exon 5 of the DRB1,3,4,5 genes. Amplified products were processed using Ion Torrent library kit. Final library pools for 30–35 samples were sequenced using Ion S5 XL, and were analyzed by HLA Typestream 1.1. Results Results of typing 163 samples showed an overall % concordance of 99.7% with SBT reference typing. An average read depth for the key exons was 354 with the maximum of 598 and the minimum of 152. 4.8% of the total 2899 allele assignments had allele imbalance of 30% or less; however, the reads for typing were above 2000 to provide enough coverage without dropout. Conclusions NGS-based HLA typing has much potential to replace routine clinical SBT typing with its power to provide allelic level typing for the 11 clinically relevant HLA loci at a single pass, with an unprecedented sequence coverage, streamlined workflow, and overall low cost. We have demonstrated for the first time that all 11 loci can be amplified in a single PCR reaction. TypeStream analysis software correctly assigned all the alleles at high percent concordance. The current formulation still has room for improvement to achieve better exon coverage and allele balance. Our approach to multiplex 11 HLA loci in a single tube offers a breakthrough for NGS typing within two days. Download : Download high-res image (90KB) Download : Download full-size image

51 - Implementation of a New Automated Sample Quality Control Tool in a Whole Exome Sequencing Workflow

Objectives: The German Cancer Research Center (DKFZ) is one of the largest biomedical research institutions in Germany. The High Throughput Sequencing Unit of the DKFZ Genomics and Proteomics Core Facility provides sequencing services for multiple applications. This project demonstrates the use of an automated electrophoresis system as a quality control (QC) tool in a whole exome sequencing workflow. Mandatory for the experimental success of whole exome sequencing is the quality of the incoming genomic DNA (gDNA) material and the DNA samples at various stages of the library preparation workflow. Methodology: Exome libraries were prepared according to the Agilent Low Input Sure-SelectXT Human All Exon v5 protocol from FFPE tumor tissue samples. The libraries were equimolar pooled. Each pool was sequenced on two lanes using the Illumina HiSeq 4000 System with 100 bp paired end sequencing. To ensure success quality control was verified with an Agilent 4200 TapeStation system of the received gDNA samples and during the library preparation. Results: Intermediate QC steps were taken throughout the protocol to monitor library preparation for sequencing, such as evaluation of DNA after fragmentation, analysis of adapter-ligated and amplified DNA, and lastly, qualification of the final library. The initial QC of incoming gDNA was determined based on the DNA integrity number (DIN). All samples had a low DNA integrity, what is usual for DNA extracted from FPPE material. Due to the low quality of the DNA material, a modified fragmentation protocol was used. Modification of the fragmentation enabled to obtain meaningful sequencing results. Conclusion: Quality control is an important part of NGS workflows, library preparation protocols recommend quantification and qualification of the DNA samples at various stages. The increasing sample throughput creates a need for automation especially in a core facility where many precious samples are proceeded with time pressure. The implementation of the automated electrophoresis system in the whole exome sequencing workflow enabled to increase the efficiency of the workflow and ensure good sequencing results.

A genetic algorithm for multigroup energy structure search

The generation of multigroup neutron cross-section libraries is a key issue of the multigroup transport calculations in reactor physics. The correct choice of the boundaries of the energy groups, in particular, is decisive for obtaining reliable results. Knowledge of the reactor physics, general and specific of the studied reactor, along with long and refined analyses are required for finding out a reasonable energy structure, which is specific for the considered reactor and might be unsuitable for other systems. The genetic algorithm presented in this work aims to choose the most appropriate energy structure for the considered system to collapse a fine multigroup library into a few-groups one, usable for transient transport calculations. The user is free to choose the number of energy groups of the final library, which is in direct relation with the precision required and the time available for the simulation. The methodology is coupled with SIMMER-III code and applied to 3 reactor systems: ESNII+ ASTRID, ESFR and MSFR. The results show that the algorithm can find representative energy structures, providing accurate results on the multiplication factor. The results of each test are analyzed, showing how different compositions, geometries and neutron spectra guide the algorithm choices, so demonstrating the effectiveness of the method.

Systematic review and meta-analyses of foodservice interventions and their effect on nutritional outcomes and satisfaction of adult oncology patients.

AimAn understanding of effective foodservice interventions on nutrition outcomes in adult patients with cancer is required to support clinical decision making. This systematic review aimed to determine the effect of foodservice interventions across a range of nutritional outcomes and satisfaction of hospitalised and ambulatory adult oncology patients.MethodsThe review protocol was registered with PROSPERO (CRD42016045772). Six databases were searched using search terms associated with the intervention and population. No date or language restrictions were applied. Authors applied the inclusion criteria to titles and abstracts and then full‐text papers. The final library was assessed for risk of bias. Outcome data were combined narratively and, where possible, by meta‐analysis.ResultsFrom the title and abstract review of 4414 studies, 12 studies testing the effect of foodservice interventions were included in this review. Meta‐analyses demonstrated significantly greater energy (mean difference 1.54 MJ/day; 95% CI 0.85–2.23 MJ/day) and protein (mean difference 18.98 g/day; 95% CI 11.58–26.39 g/day) intake through the addition of oral nutrition supplements. Other positive effects on anthropometric outcomes were also recorded. Patient satisfaction was enhanced through other foodservice interventions.ConclusionsLimited original research was found exploring the effect of foodservice interventions in oncology patients. Significant findings were found in favour of the intervention across a range of nutritional outcomes, suggesting that foodservice interventions may improve clinical outcomes and satisfaction in this population. Effective foodservice interventions for oncology patients remain under‐researched, so we encourage dietitians and foodservice staff to implement rigorous study designs to evaluate and publish interventions in this clinical group.

Automation of PacBio SMRTbell NGS library preparation for bacterial genome sequencing

BackgroundThe PacBio RS II provides for single molecule, real-time DNA technology to sequence genomes and detect DNA modifications. The starting point for high-quality sequence production is high molecular weight genomic DNA. To automate the library preparation process, there must be high-throughput methods in place to assess the genomic DNA, to ensure the size and amounts of the sheared DNA fragments and final library.FindingsThe library construction automation was accomplished using the Agilent NGS workstation with Bravo accessories for heating, shaking, cooling, and magnetic bead manipulations for template purification.The quality control methods from gDNA input to final library using the Agilent Bioanalyzer System and Agilent TapeStation System were evaluated.ConclusionsAutomated protocols of PacBio 10 kb library preparation produced libraries with similar technical performance to those generated manually. The TapeStation System proved to be a reliable method that could be used in a 96-well plate format to QC the DNA equivalent to the standard Bioanalyzer System results. The DNA Integrity Number that is calculated in the TapeStation System software upon analysis of genomic DNA is quite helpful to assure that the starting genomic DNA is not degraded. In this respect, the gDNA assay on the TapeStation System is preferable to the DNA 12000 assay on the Bioanalyzer System, which cannot run genomic DNA, nor can the Bioanalyzer work directly from the 96-well plates.

Extensive libraries of gene truncation variants generated by in vitro transposition.

The detailed analysis of the impact of deletions on proteins or nucleic acids can reveal important functional regions and lead to variants with improved macromolecular properties. We present a method to generate large libraries of mutants with deletions of varying length that are randomly distributed throughout a given gene. This technique facilitates the identification of crucial sequence regions in nucleic acids or proteins. The approach utilizes in vitro transposition to generate 5΄ and 3΄ fragment sub-libraries of a given gene, which are then randomly recombined to yield a final library comprising both terminal and internal deletions. The method is easy to implement and can generate libraries in three to four days. We used this approach to produce a library of >9000 random deletion mutants of an artificial RNA ligase enzyme representing 32% of all possible deletions. The quality of the library was assessed by next-generation sequencing and detailed bioinformatics analysis. Finally, we subjected this library to in vitro selection and obtained fully functional variants with deletions of up to 18 amino acids of the parental enzyme that had been 95 amino acids in length.

The Desired Length of the Library Insert was Influenced by the Degree of mRNA Purification in Poplar

The typical workflow of an RNA-seq assay involves the extraction and further purification of mRNA. The size of the target DNA fragments in the final library is a key parameter for RNA-Seq library construction. In our experiment, we found that fragmentation is influenced by the purification of mRNA and leads to different insert sizes in the transcriptome libraries. This study compared many purification methods after extraction mRNA using magnetic silica beads. To assess the quality of the mRNA obtained from these options, the size of library was analyzed on the Agilent 2100 Bioanalyzer to measure whether the library is constructed successfully. Results of the best purification method could thoroughly remove the rRNA, tRNA and other impurities to obtain complete, high-purity mRNA molecules. The discovery of this phenomenon, the summary of the rules and the related purification reagents ratio all these can help us further exploited RNA-seq protocols.