scholarly journals SALP, a new single-stranded DNA library preparation method especially useful for the high-throughput characterization of chromatin openness states

BMC Genomics ◽  
2018 ◽  
Vol 19 (1) ◽  
Author(s):  
Jian Wu ◽  
Wei Dai ◽  
Lin Wu ◽  
Jinke Wang
Keyword(s):  
High Throughput ◽  
Preparation Method ◽  
Library Preparation ◽  
Single Stranded Dna ◽  
Dna Library ◽  
Library Preparation Method

scholarly journals SALP, a new single-stranded DNA library preparation method especially useful for the high-throughput characterization of chromatin openness states

2017 ◽  
Author(s):  
Jian Wu ◽  
Wei Dai ◽  
Ling Wu ◽  
Jinke Wang
Keyword(s):  
Biological Sciences ◽  
High Throughput ◽  
High Sensitivity ◽  
Single Strand ◽  
Library Preparation ◽  
Single Stranded Dna ◽  
Dna Library ◽  
Next Generation Sequencing Ngs ◽  
Multiple Cells

AbstractBased on a novel kind of single strand adaptor (SSA), this study developed a new method to construct next-generation sequencing (NGS) library, named as SALP, representing Single strand Adaptor Library Preparation. The key creativity of the method lies in the design and verification of a special adaptor that can be efficiently linked to the 3′ end of single-stranded DNA, which is a double-stranded oligonucleotide with a 3′ overhang of 3 random nucleotides. This method can start with the denatured DNAs or chromatins fragmented by different methods such as Tn5 tagmentation, enzyme digestion and sonication. When applied to Tn5-tagmented chromatin, SALP overcomes the key limitation of the current ATAC-seq method and develops a high-throughput NGS library construction and sequencing approach, SALP-seq, which can be used to comparatively characterize the chromatin openness state of multiple cells simply and unbiasly. In this way, the comparative chromatin openness states of four different cell lines, including GM12878, HepG2, HeLa and 293T, were successfully characterized. This study also demonstrated that SALP-seq could characterize the chromatin openness states with 105 to 500 cells, indicating the high sensitivity of SALP-seq in characterizing chromatin state of cells. SALP should have wide applications in the future biological sciences and biomedicine.

Novel target capture DNA library preparation method using CircLigase-mediated hook ligation

2020 ◽  
Vol 59 ◽  
pp. 44-50
Author(s):  
Han Ren ◽  
Yang Xi ◽  
Zhanqing Li ◽  
Dengwei Zhang ◽  
Fubaoqian Huang ◽  
...  
Keyword(s):  
Preparation Method ◽  
Library Preparation ◽  
Dna Library ◽  
Target Capture ◽  
Novel Target ◽  
Library Preparation Method

scholarly journals High throughput barcoding method for genome-scale phasing

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
David Redin ◽  
Tobias Frick ◽  
Hooman Aghelpasand ◽  
Max Käller ◽  
Erik Borgström ◽  
...  
Keyword(s):  
High Throughput ◽  
Preparation Method ◽  
Library Preparation ◽  
Structural Variants ◽  
Human Genomics ◽  
Short Read Sequencing ◽  
Base Calling ◽  
Cost Efficient ◽  
Genome Scale ◽  
Library Preparation Method

AbstractThe future of human genomics is one that seeks to resolve the entirety of genetic variation through sequencing. The prospect of utilizing genomics for medical purposes require cost-efficient and accurate base calling, long-range haplotyping capability, and reliable calling of structural variants. Short-read sequencing has lead the development towards such a future but has struggled to meet the latter two of these needs. To address this limitation, we developed a technology that preserves the molecular origin of short sequencing reads, with an insignificant increase to sequencing costs. We demonstrate a novel library preparation method for high throughput barcoding of short reads where millions of random barcodes can be used to reconstruct megabase-scale phase blocks.

scholarly journals High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity

mSystems ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Jeremiah J. Minich ◽  
Greg Humphrey ◽  
Rodolfo A. S. Benitez ◽  
Jon Sanders ◽  
Austin Swafford ◽  
...  
Keyword(s):  
16S Rrna ◽  
Microbial Ecology ◽  
High Throughput ◽  
Preparation Method ◽  
Library Preparation ◽  
Reaction Volume ◽  
Liquid Handling ◽  
Amplicon Library ◽  
The Cost ◽  
Library Preparation Method

ABSTRACT Next-generation sequencing technologies have enabled many advances across biology, with microbial ecology benefiting primarily through expanded sample sizes. Although the cost of running sequencing instruments has decreased substantially over time, the price of library preparation methods has largely remained unchanged. In this study, we developed a low-cost miniaturized (5-µl volume) high-throughput (384-sample) amplicon library preparation method with the Echo 550 acoustic liquid handler. Our method reduces costs of library preparation to $1.42 per sample, a 58% reduction compared to existing automated methods and a 21-fold reduction from commercial kits, without compromising sequencing success or distorting the microbial community composition analysis. We further validated the optimized method by sampling five body sites from 46 Pacific chub mackerel fish caught across 16 sampling events over seven months from the Scripps Institution of Oceanography pier in La Jolla, CA. Fish microbiome samples were processed with the miniaturized 5-µl reaction volume with 0.2 µl of genomic DNA (gDNA) and the standard 25-µl reaction volume with 1 µl of gDNA. Between the two methods, alpha diversity was highly correlated (R2 > 0.95), while distances of technical replicates were much lower than within-body-site variation (P < 0.0001), further validating the method. The cost savings of implementing the miniaturized library preparation (going from triplicate 25-µl reactions to triplicate 5-µl reactions) are large enough to cover a MiSeq sequencing run for 768 samples while preserving accurate microbiome measurements. IMPORTANCE Reduced costs of sequencing have tremendously impacted the field of microbial ecology, allowing scientists to design more studies with larger sample sizes that often exceed 10,000 samples. Library preparation costs have not kept pace with sequencing prices, although automated liquid handling robots provide a unique opportunity to bridge this gap while also decreasing human error. Here, we take advantage of an acoustic liquid handling robot to develop a high-throughput miniaturized library preparation method of a highly cited and broadly used 16S rRNA gene amplicon reaction. We evaluate the potential negative effects of reducing the PCR volume along with varying the amount of gDNA going into the reaction. Our optimized method reduces sample-processing costs while continuing to generate a high-quality microbiome readout that is indistinguishable from the original method.

scholarly journals Biases in the SMART-DNA library preparation method associated with genomic poly dA/dT sequences

PLoS ONE ◽  
2017 ◽  
Vol 12 (2) ◽  
pp. e0172769 ◽  
Author(s):  
Oriya Vardi ◽  
Inbal Shamir ◽  
Elisheva Javasky ◽  
Alon Goren ◽  
Itamar Simon
Keyword(s):  
Preparation Method ◽  
Library Preparation ◽  
Dna Library ◽  
Library Preparation Method

scholarly journals Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma

2015 ◽  
Author(s):  
Philip Burnham ◽  
Min Seong Kim ◽  
Sean Agbor-Enoh ◽  
Helen Luikart ◽  
Hannah A Valantine ◽  
...  
Keyword(s):  
Lung Transplant ◽  
Preparation Method ◽  
Fragmentation Pattern ◽  
Library Preparation ◽  
Transplant Recipients ◽  
Cell Free Dna ◽  
Single Stranded Dna ◽  
Free Dna ◽  
Lung Transplant Recipients ◽  
Library Preparation Method

Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of cfDNA in blood, can be readily adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparation, however, is insensitive to ultrashort, degraded and single-stranded cfDNA. Drawing inspiration from recent technical advances in ancient genome analyses, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in the plasma of lung transplant recipients (40 samples, six patients). We found that the ssDNA library preparation yields a greater portion of sub-100 bp DNA, as well as an increased relative abundance of human mitochondrial cfDNA (10.7x) and microbial cfDNA (71.3x). We report the fragmentation pattern of mitochondrial, nuclear genomic and microbial cfDNA over a broad fragment length range. We furthermore report the first observation of donor-specific mitochondrial cfDNA in the circulation of lung transplant recipients. We found that donor-specific mitochondrial cfDNA molecules are significantly shorter than those specific to the recipient. The higher yield of viral, microbial and fungal sequences that result from the single-stranded ligation approach reduces the cost and increase the sensitivity of cfDNA-based monitoring for infectious complications after transplantation. An ssDNA library preparation method provides a more informative window into understudied forms of cfDNA, including mitochondrial and microbial derived cfDNA and short fragment nuclear genomic cfDNA, while retaining information provided by standard dsDNA library preparation methods.

scholarly journals Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries

2021 ◽  
Author(s):  
Terence S. Crofts ◽  
Alexander G. McFarland ◽  
Erica M. Hartmann
Keyword(s):  
Amino Acid ◽  
High Throughput ◽  
Preparation Method ◽  
Metagenomic Library ◽  
Amino Acid Identity ◽  
Library Preparation ◽  
Novel Genes ◽  
Acid Identity ◽  
Metagenomic Libraries ◽  
Library Preparation Method

ABSTRACTFunctional metagenomic libraries, physical bacterial libraries which allow the high-throughput capture and expression of microbiome genes, have been instrumental in the sequence-naïve and cultivation-independent discovery of novel genes from microbial communities. Preparation of these libraries is limited by their high DNA input requirement and their low cloning efficiency. Here, we describe a new method, METa assembly, for extremely efficient functional metagenomic library preparation. We apply tagmentation to metagenomic DNA from soil and gut microbiomes to prepare DNA inserts for high-throughput cloning into functional metagenomic libraries. The presence of mosaic end sequences in the resulting DNA fragments synergizes with homology-based assembly cloning to result in a 300-fold increase in library size compared to traditional blunt cloning based protocols. Compared to published libraries prepared by state-of-the-art protocols we show that METa assembly is on average 23- to 270-fold more efficient and can be effectively used to prepare gigabase-sized libraries with as little as 200 ng of input DNA. We demonstrate the utility of METa assembly to capture novel genes based on their function by discovering novel aminoglycoside (26% amino acid identity) and colistin (36% amino acid identity) resistance genes in soil and goose gut microbiomes. METa assembly provides a streamlined, flexible, and efficient method for preparing functional metagenomic libraries, enabling new avenues of genetic and biochemical research into low biomass or scarce microbiomes.IMPORTANCEMedically and industrially important genes can be recovered from microbial communities by high-throughput sequencing but are limited to previously sequenced genes and their relatives. Cloning a metagenome en masse into an expression host to produce a functional metagenomic library is a sequence-naïve and cultivation-independent method to discover novel genes. This directly connects genes to functions, but the process of preparing these libraries is DNA greedy and inefficient. Here we describe a library preparation method that is an order of magnitude more efficient and less DNA greedy. This method is consistently efficient across libraries prepared from cultures, a soil microbiome, and from a goose fecal microbiome and allowed us to discover novel antibiotic resistance genes. This new library preparation method will potentially allow for the functional metagenomic exploration of microbiomes that were previously off limits due to their rarity or low microbial biomass, such biomedical swabs or exotic samples.

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