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PubMed2026

Comparative metagenomic assessment of Illumina-compatible library preparation methods, short-read lengths, and PacBio HiFi sequencing reveals differences in microbial and functional diversity recovery from a complex environmental sample.

UNLABELLED: Metagenomics enables comprehensive exploration of microbial communities but is influenced by library preparation and sequencing technologies, affecting recovery of microbial genomes and proteins. Here, we benchmarked six Illumina-compatible short-read library preparation conditions in triplicate at 2 × 150 bp and 2 × 250 bp read lengths alongside PacBio HiFi long-read sequencing using a composite environmental sample of marine mangrove sediment and terrestrial palm tree soil. Longer short reads (2 × 250 bp) combined with optimal library preparation approaches improved assembly quality, protein detection, and metagenome-assembled genome (MAG) recovery, achieving results approaching those of long-read sequencing. TruSeq libraries at 2 × 250 bp recovered more than sevenfold more unique proteins than the same kit at 2 × 150 bp (811,701 vs 110,108) using the same number of sequencing reads, while recovering a comparable number of high-quality MAGs to PacBio HiFi long-read sequencing (11 vs 18) and surpassing it in protein discovery by almost 10-fold (811,701 vs 87,745) at less than half of the sequencing cost. Furthermore, biosynthetic gene cluster analysis identified 46 biosynthetic gene clusters in TruSeq-250PE assemblies compared to 38 in PacBio HiFi, with several showing no close match in the MIBiG database. Although long reads yield more contiguity and complete genomes, longer short reads offer a cost-effective, scalable alternative for uncovering microbial and functional diversity. These findings provide critical guidance for metagenomic experimental design, demonstrating that strategic selection of library preparation chemistry and sequencing parameters can reveal more unknown microbial information in complex biomes without requiring additional sequencing depth. IMPORTANCE: Metagenomic outcomes are strongly influenced by library preparation and sequencing strategies, yet their combined effects in complex environmental samples remain poorly defined. Here, we provide the first direct comparison of Illumina NovaSeq short-read metagenomic sequencing at 2 × 150 bp and 2 × 250 bp across multiple library preparation kits, alongside PacBio HiFi long-read sequencing. We show that sequencing read length and library preparation critically shape assembly quality, protein recovery, and metagenome-assembled genome (MAG) reconstruction. These findings demonstrate that short-read sequencing at 2 × 250 bp, with appropriate library preparation, can match long-read technologies in MAG recovery while substantially surpassing them in protein discovery. With less than half of the sequencing price and a 3.5-fold reduction in cost per gigabase of usable data, this method facilitates more accessible large-scale metagenomic analysis within complex environmental systems.

Metagenomics↗

PubMed2024

High-throughput DNA extraction and cost-effective miniaturized metagenome and amplicon library preparation of soil samples for DNA sequencing.

Reductions in sequencing costs have enabled widespread use of shotgun metagenomics and amplicon sequencing, which have drastically improved our understanding of the microbial world. However, large sequencing projects are now hampered by the cost of library preparation and low sample throughput, comparatively to the actual sequencing costs. Here, we benchmarked three high-throughput DNA extraction methods: ZymoBIOMICS™ 96 MagBead DNA Kit, MP BiomedicalsTM FastDNATM-96 Soil Microbe DNA Kit, and DNeasy® 96 PowerSoil® Pro QIAcube® HT Kit. The DNA extractions were evaluated based on length, quality, quantity, and the observed microbial community across five diverse soil types. DNA extraction of all soil types was successful for all kits, however DNeasy® 96 PowerSoil® Pro QIAcube® HT Kit excelled across all performance parameters. We further used the nanoliter dispensing system I.DOT One to miniaturize Illumina amplicon and metagenomic library preparation volumes by a factor of 5 and 10, respectively, with no significant impact on the observed microbial communities. With these protocols, DNA extraction, metagenomic, or amplicon library preparation for one 96-well plate are approx. 3, 5, and 6 hours, respectively. Furthermore, the miniaturization of amplicon and metagenome library preparation reduces the chemical and plastic costs from 5.0 to 3.6 and 59 to 7.3 USD pr. sample. This enhanced efficiency and cost-effectiveness will enable researchers to undertake studies with greater sample sizes and diversity, thereby providing a richer, more detailed view of microbial communities and their dynamics.

Metagenome↗

PubMed2002

The use of a supported base and strong cation exchange (SCX) chromatography to prepare a variety of structurally-diverse molecular libraries prepared by solution-phase methods.

The preparation of molecular libraries of aminomethylbiaryls, allylic amines, and ethanolamines using solution-phase methodology is described. In particular, the use of a solid-supported base reagent (PTBD resin) and strong cation exchange(SCX) resin to effect 'catch and release' purification across these diverse libraries is highlighted.

Alkylation↗

PubMed2003

An unbiased cDNA library prepared from isolated Aplysia sensory neuron processes is enriched for cytoskeletal and translational mRNAs.

Local protein synthesis is required for long-lasting synapse-specific plasticity in cultured Aplysia sensorimotor synapses. To identify synaptically localized mRNAs, we prepared a cDNA library from isolated sensory neurites. By sequence analysis, we estimate that the library contains 263 distinct mRNAs, with 98 of these mRNAs constituting 70% of all clones. The localized transcripts are enriched for mRNAs encoding cytoskeletal elements and components of the translational machinery. In situ hybridization confirms that the mRNAs for at least eight of these transcripts are present in distal neurites. Immunocytochemistry reveals that serotonin regulates the translation of one of the localized mRNAs, that encoding alpha1-tubulin. Our identification of mRNAs encoding cytoskeletal elements suggests that local protein synthesis is required for the growth of new synaptic connections associated with persistent synaptic strengthening. Our finding of mRNAs encoding components of the translational machinery suggests that local protein synthesis serves to increase the translational capacity of synapses.

Animals↗

PubMedDate not supplied

Library preparation of derivatives of 1,4,10,13-tetraoxa-7,16-diaza-cycloctadecane and their fluorescence behavior for signaling purposes.

In the present work, we report the library preparation on solid supports of 20 derivatives of 1,4,10,13-tetraoxa-7,15-diaza-cycloctadecane 1(a-e)(w,x,y,z) carrying a fluorescent dansyl group. The sensing fluorescence behavior of these materials toward alkali and alkali earth metal ions was studied by packing the beads into a conventional flow-through cell in a FIA (flow injection analysis) approach. The fluorescence emission of these materials' responses shows a fluorescence increase to Li+, Na+, K+, NH4+, Ca2+, and Mg2+ with maximum sensitivity for Mg2+ over the rest of the ions. The analytical potential of these materials is outlined, and the sensing response mechanism, based on a photoinduced electron-transfer process, is proposed.

Alkanes↗

PubMed1987

Predefined gene transfer for expression of a glycosphingolipid antigen by transfection with a cosmid genomic library prepared from a cell line in which the specific glycosphingolipid is highly expressed.

The deliberate transfer of globotriaosylceramide (Gb3) expression in mouse lymphoma L5178 cells was achieved by transfection with a cosmid DNA library prepared from human Burkitt lymphoma Ramos cells in which Gb3 was highly expressed. The recipient mouse lymphoma cells did not contain Gb3 but did contain its direct precursor, lactosylceramide. The transfected cells expressed Gb3, detected both chemically and immunologically, and contained human DNA detected by an Alu sequence probe. This model demonstrates a general method for studying glycosyltransferase genes and other factors necessary for the expression of glycosphingolipid antigens.

Animals↗

PubMed2000

Cloning the antibody response in humans with chronic inflammatory disease: immunopanning of subacute sclerosing panencephalitis (SSPE) brain sections with antibody phage libraries prepared from SSPE brain enriches for antibody recognizing measles virus antigens in situ.

In central nervous system (CNS) infectious and inflammatory diseases of known cause, oligoclonal bands represent antibody directed against the causative agent. To determine whether disease-relevant antibodies can be cloned from diseased brain, we prepared an antibody phage display library from the brain of a human with subacute sclerosing panencephalitis (SSPE), a chronic encephalitis caused by measles virus, and selected the library against SSPE brain sections. Antibodies that were retrieved reacted strongly with measles virus cell extracts by enzyme-linked immunosorbent assay and were specific for the measles virus nucleocapsid protein. These antibodies immunostained cells in different SSPE brains but not in control brain. Our data provide the first demonstration that diseased brain can be used to select in situ for antibodies directed against the causative agent of disease and point to the potential usefulness of this approach in identifying relevant antibodies in chronic CNS or systemic inflammatory diseases of unknown cause.

Antibodies, Viral↗

PubMed1994

Human monoclonal Fab fragments from a combinatorial library prepared from an individual with a low serum titer to a virus.

An IgG1k lambda Fab library was generated on the surface of phage beginning with bone marrow RNA from a healthy 22-year-old human donor. The donor had been immunized to measles in his early childhood but had only a low serum titer to a measles antigen preparation. The resulting library of approximately 10(7) clones was panned against the measles antigen preparation and three positive Fab-producing clones identified by ELISA. One of the Fabs was found to be specific to measles and to bind with high apparent affinity (10(8) M-1). The other two bind with lower affinity and show marked cross-reactivity with a number of other antigens. They possess heavy chains derived, with extensive somatic modification, from the single member gene family VH6. The study indicates that both high-affinity specific antibodies and lower affinity polyreactive antibodies can be derived from the library approach under appropriate conditions.

Adult↗

PubMed1991

Isolation of wound-specific cDNA clones from a cDNA library prepared with mRNAs of alkali-burned rabbit corneas.

Alkali burn is one of the most severe corneal injuries. In order to gain a better understanding of the healing of alkali-burned corneas, it is necessary to identify and characterize proteins that are specifically synthesized by the injured corneal tissues. In this study, we developed a useful procedure to identify and isolate cDNA clones that encode messenger ribonucleic acids (mRNAs) that are specific and/or abundant in alkali-burned rabbit corneas (ARCs), but absent in normal rabbit corneas (NRCs). At first, a cDNA library was prepared by cloning cDNA of mRNA isolated from ARCs into the lambda ORF-8 vector. A differential plaque hybridization was used to screen 2.5 x 10(4) plaque-forming units (pfu) from an ARC cDNA library using 32P-labeled cDNAs prepared from mRNA of ARCs and NRCs. Thirty-seven cDNA clones of mRNAs specific for ARCs were identified and isolated in their pureform. The cDNA inserts of these lambda ORF-8 phages were subcloned into the pSM216 vector by in vivo recombination. The cDNA inserts then were characterized by restriction enzyme digestion, i.e., BamHI, HindIII, and EcoRI. The size of the cDNA inserts ranged from 210 to 5,000 base pairs. Using Northern blot hybridization of total RNA prepared from polymorphonuclear neutrophils, mononuclear leukocytes, alkali-burned corneas, and normal corneas, the cDNA clones were divided into three groups. Five cDNA clones encoded mRNA of corneal cells in ARCs. Twenty-four cDNA clones derived from mRNA of inflammatory cells were present in alkali-burned corneas, but Northern blot hybridization failed to identify mRNA of discrete sizes.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkalies↗

PubMed2007

Combinatorial approach for ferroelectric material libraries prepared by liquid source misted chemical deposition method.

Combinatorial approach for discovering novel functional materials in the huge diversity of chemical composition and processing conditions has become more important for breakthrough in thin film electronic and energy-conversion devices. The efficiency of combinatorial method depends on the preparation of a reliable high-density composition thin-film library. The physico-chemical properties of each sample on the library should be similar to those of the corresponding samples prepared by one-by-one conventional methods. We successfully developed the combinatorial liquid source misted chemical deposition (LSMCD) method and demonstrated its validity in screening the chemical composition of Bi3.75LaxCe0.25-xTi3O12 (BLCT) for high remanent polarization (Pr). LSMCD is a cheap promising combinatorial screening tool. It can control the composition up to ppm level and produce homogeneous multicomponent library. LSMCD method allows us to prepare BLCT thin-film library at the variation of 0.4 mol% of La. Maximum 2Pr is 35 microC/cm-2 at x=0.21. The intensity of (117) XRD peak is quantitatively related to 2Pr. Newly developed scanning piezoelectric deformation measurement for nano-sized samples using scanning probe microscope (SPM) is also found out to be reliable for determining the relative ranking of Pr value rapidly.

Journal Article↗

PubMed2003

Antioxidative properties of tripeptide libraries prepared by the combinatorial chemistry.

Two series of combinatorial tripeptide libraries were constructed, based on an antioxidative peptide isolated from a soybean protein hydrolysate. One was a library of 108 peptides containing either His or Tyr residues. Another was a library of 114 peptides related to Pro-His-His, which had been identified as an active core of the antioxidative peptide. The antioxidative properties of these libraries were examined by several methods, such as the antioxidative activity against the peroxidation of linoleic acid, the reducing activity, the radical scavenging activity, and the peroxynitrite scavenging activity. Two Tyr-containg tripeptides showed higher activities than those of two His-containing tripeptides in the peroxidation of linoleic acid. Tyr-His-Tyr showed a strong synergistic effects with phenolic antioxidants. However, the tripeptide had only marginal reducing activity and a moderate peroxynitrite scavenging activity. Cysteine-containing tripeptides showed the strong peroxynitrite scavenging activity. Change of either the N-terminus or C-terminus of Pro-His-His to other amino acid residues did not significantly alter their antioxidative activity. Tripeptides containing Trp or Tyr residues at the C-terminus had strong radical scavenging activities, but very weak peroxynitrite scavenging activity. The present results allow us to understand why protein digests have such a variety of antioxidative properties.

Antioxidants↗

PubMed1993

Selection of an active single chain Fv antibody from a protein linker library prepared by enzymatic inverse PCR.

Enzymatic inverse PCR mutagenesis was developed as a simple and reliable method for the construction of large libraries of site-directed mutants. Enzymatic inverse PCR library mutagenesis uses a single PCR fragment and is restriction-site independent. The usefulness of the technique was demonstrated by the design of a single chain linker for an antibody Fv fragment without computer modeling. The Fv fragment of an antibody specific for a metal chelate was expressed in active form in the periplasm of E. coli. The light and the heavy chains of the Fv are expressed as a bicistronic mRNA. Enzymatic inverse PCR mutagenesis was used to construct a library of 3 x 10(5) Fv mutants, in which the C-terminus of the light chain was connected to the N-terminus of the heavy chain by a 15-amino acid peptide linker of variable composition. After plating, active mutant colonies were identified by screening colony filter lifts with a radiolabeled hapten, N'-(2-hydroxyethyl)-p-thioureidobenzyl EDTA. About 0.2% of the mutants were positive, and a selected sFv clone was shown to have the same affinity as the Fv (9 x 10(9)) and was similar to the whole antibody (11 x 10(9)). This example compares favorably with both of the other approaches to constructing sFv's; namely, molecularly modeled linkers as well as universal linkers, which have often yielded significantly lower affinities than whole antibodies or Fabs. The enzymatic inverse PCR library mutagenesis approach is simple and reliable and can be used to obtain linkers for the great majority of antibodies for which no structural data are available. More generally, it can be used to modify DNA coding for any structural protein or regulatory element.

Base Sequence↗

PubMed1982

Representation of DNA sequences in recombinant DNA libraries prepared by restriction enzyme partial digestion.

We present a theoretical study of the fraction of sequences incorporated in a recombinant DNA partial digest library as a function of the size of the library. The fraction incorporated depends on the degree of restriction enzyme partial digestion. If all restriction sites in the target DNA can be cleaved with the same rate, optimum incorporation of sequences is observed when the number average length of the digested DNA equals the desired average length of the cloned insert. Overdigestion severely reduces the fraction of sequences present in a sample of clones. Heterogeneity in restriction enzyme cleavage rates also reduces the fraction incorporated, and underdigestion improves sequence representation in the face of cleavage rate heterogeneity. Practical methods for determining the number average length of partially digested DNAs are also presented.

Base Composition↗

PubMed2006

Construction and characterization of a highly redundant Pseudomonas aeruginosa genomic library prepared from 12 clinical isolates: application to studies of gene distribution among populations.

OBJECTIVE: To create, array, and characterize a pooled, high-coverage, genomic library composed of multiple biofilm-forming clinical strains of the opportunistic pathogen, Pseudomonas aeruginosa (PA). Twelve strains were obtained from patients with otorrhea, otitis media, and cystic fibrosis as a resource for investigating: difference in the transcriptomes of planktonic and biofilm envirovars; the size of the PA supragenome and determining the number of virulence genes available at the population level; and the distributed genome hypothesis. METHODS: High molecular weight genomic DNAs from 12 clinical PA strains were individually hydrodynamically sheared to produce mean fragment sizes of approximately 1.5 kb. Equimolar amounts of the 12 sheared genomic DNAs were then pooled and used in the construction of a genomic library with approximately 250,000 clones that was arrayed and subjected to quality control analyses. RESULTS: Restriction endonuclease and sequence analyses of 686 clones picked at random from the library demonstrated that >75% of the clones contained inserts larger than 0.5 kb with the desired mean insert size of 1.4 kb. Thus, this library provides better than 4.5x coverage for each of the genomes from the 12 components clinical PA isolates. Our sequencing effort ( approximately 1 million nucleotides to date) reveals that 13% of the clones present in this library are not represented in the genome of the reference P. aeruginosa strain PA01. CONCLUSIONS: Our data suggests that reliance on a single laboratory strain, such as PA01, as being representative of a pathogenic bacterial species will fail to identify many important genes, and that to obtain a complete picture of complex phenomena, including bacterial pathogenesis and the genetics of biofilm development will require characterization of the P. aeruginosa population-based supra-genome.

Child, Preschool↗

PubMed1994

New York University Dental Center Library. Preparing to meet the information needs of the dental team today and in the 21st century.

The introduction of new computer technology has been changing the way libraries provide access to the vast amount of information being produced in the fields of medicine and dentistry. The NYU Dental Library is responding to the challenge with new systems that improve its daily operation and expand users' abilities to gain access to the data they need.

Computer Communication Networks↗

PubMed1994

Gene mapping from a bovine 1;29 DNA library prepared with chromosome microdissection.

Bovine gene mapping is progressing rapidly using syntenic group mapping based on somatic cell hybrids and linkage, and to a lesser extent on in situ hybridization. Single chromosome DNA libraries are a logical next step, and this was, therefore, the aim of our laboratory. Since we have access to several cattle with t(1;29) and this chromosome is readily distinguishable, we chose this as our first target--recognizing that we would not produce a "single" chromosome library in the strict sense because two autosomes are represented. We utilized an inverted microscope and a micromanipulator fitted with glass instruments pulled specifically to dissect off approximately 100 t(1;29) chromosomes per microdrop. A glass chamber made to accommodate a hanging drop was used to extract the DNA under a dissecting microscope. The DNA was then cleaved with EcoRI and inserted in lambda gtwes arms. Host cells were then infected with these phage and positive clones obtained. The first clone, isolated from this library by hybridization with a human collagen 6A1 cDNA, was mapped by in situ hybridization to bovine Chromosome some (Chr) 1q12-q14, near the centromere. The second clone, an anonymous DNA fragment (D1S11), was mapped to 1q43-q46, near the terminal end.

Animals↗

PubMed1995

The construction and analysis of M13 libraries prepared from YAC DNA.

Yeast artificial chromosomes (YACs) provide a powerful way to isolate and map large regions of genomic DNA and their use in genome analysis is now extensive. We modified a series of procedures to produce high quality shotgun libraries from small amounts of YAC DNA. Clones from several different libraries have been sequenced and analyzed for distribution, sequence integrity and degree of contamination from yeast DNA. We describe these procedures and analyses and show that sequencing at about 1-fold coverage, followed by database comparison (survey sequencing) offers a relatively quick method to determine the nature of previously uncharacterized cosmid or YAC clones.

Bacteriophage M13↗

PubMedDate not supplied

Identification of synthetic phosphatidylserine translocases from a combinatorial library prepared by directed split-and-pool synthesis.

Simple sulfonamide and amide derivatives of tris(2-aminoethyl)amine (Tren) are known to promote the translocation or flip-flop of phosphatidylcholine, but not phosphatidylserine, across bilayer membranes. This paper describes the synthesis of a 300-member, spatially encoded library of Tren derivatives with appended peptide--sulfonamide and peptide--urea arms. The library was synthesized using the Encore method with SynPhase lanterns as the solid support. A high-throughput assay was developed to screen individual members of the library for an ability to translocate a fluorescent NBD derivative of phosphatidylserine across vesicle membranes. Several lead compounds were identified, and one was synthesized independently to confirm its high phosphatidylserine translocation activity.

Combinatorial Chemistry Techniques↗