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Library preparation

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Fluorescent amplification for next generation sequencing (FA-NGS) library preparation

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generation sequencing (FA-NGS) library preparation. Conventional library preparation typically requires quality control (QC) testing for individual libraries such as amplification success evaluation and quantification, none of which occur until the end of the library preparation process.. We modified two distinct library preparation workflows by replacing PCR and quantification with qPCR using SYBR Green I.

Bead-linked transposomes enable a normalization-free workflow for NGS library preparation

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The Nextera DNA Flex library preparation kit was tested on a variety of liquid handling platforms. DNA Flex Library Preparation Kit. illumina-marketing/documents/products/appnotes/nextera-dna-flex- small-genomes-application-note pdf

Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing

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Robust Sub-nanomolar Library Preparation for High Throughput Next Generation. A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input.

A ligation-based single-stranded library preparation method to analyze cell-free DNA and synthetic oligos

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Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma. https://doi.org/10.1038/srep27859.. Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase. doi.org/10.1093/nar/gkx033.. https://doi.org/10.1093/nar/gkw1110.. https://doi.org/10.4161/rna.29304..

Automation of Spatial Transcriptomics library preparation to enable rapid and robust insights into spatial organization of tissues

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We also tested the Bravo ST library preparation proto- col on real MOB and small prostate cancer needle biopsy tissue samples. The Bravo automated ST library preparation protocol thus achieves excellent sensitivity even when little input material is available. Further scalability should be possible because the Bravo system can process 96 samples simultaneously with no increase in running time.

A toolkit for studying Varroa genomics and transcriptomics: Preservation, extraction, and sequencing library preparation

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However, the num- ber of samples we processed for library preparation and sequencing resulted in high-quality mapping to the ref- erence genome, and none of the samples deviated strongly from the rest, suggesting we have a good repre- sentation of the treatments. 2 Alignment of RNA libraries from Varroa mite total RNA to V.

Systematic comparison of small RNA library preparation protocols for next-generation sequencing

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In some of the library preparation protocols (see below), the adapters included in the kits were replaced by custom adapters. Python scripts were used to trim HD adapters and also the random 3-base sequences upstream of the small RNA inserts for the SMARTer protocol. The sampling of the reads was done using seqtk, version 1.0-r31 (H. For the annotation of the short side products (10–.

A comparison between whole transcript and 3’ RNA sequencing methods using Kapa and Lexogen library preparation methods

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1 Key library preparation steps for the Trad-KAPA (left) and 3 ’ -LEXO (right) methods. The sequencing results for the Trad-KAPA and 3’-LEXO libraries were compared to determine their relative advantages and disadvantages.. We first mapped the reads to the mouse genome, and confirmed that the Trad-KAPA reads covered the whole transcript, while 3’-LEXO reads only covered the 3′.

Next-generation sequencing library construction on a surface

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Preparation of poly-acrylamide gel surface. Genomic DNA was stained with YoYo-1 (ThermoFisher) in phosphate buffered saline (pH 8.3) overnight at 4 °C.. PDMS coated coverslip was pulled up and out of the reservoir at. Surface tagmentation and library preparation.

Systematic evaluation of RNA-Seq preparation protocol performance

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The first goal of our study was to investigate confounding factors in RNA-Seq library preparation pro- tocols using three standard input kits: the TruSeq Stranded Total RNA and mRNA Library Prep Kits from Illumina, and a modified NuGEN Ovation® RNA-Seq System. Defining the properties of the data generated using these protocols may aid users in designing their future RNA-Seq strategies.

Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction

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With the broad success of the different chemistries, other aspects of library preparation, such as cost and ease of use, can be considered. While all sites were familiar with the RiboZero deple- tion kit, the majority of sites had no prior experience with the other kits. This may be due to the robustness of the specific methods or the quality of the written protocols.. While we did observe differences in the efficiency of library preparation, analytics remains a key caveat.

A comparative analysis of library prep approaches for sequencing low input translatome samples

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Comparison of mapping efficiency and duplication rate The number of reads varied widely among samples being prepared by different library preparation kits. Of the raw reads, 12.5 to 111 million reads mapped to mouse genome for input RNA samples while 9.2 to 94.6 million reads mapped for IP RNA samples (Fig.

Dual indexed library design enables compatibility of in-Drop single-cell RNAsequencing with exAMP chemistry sequencing platforms

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For the new TruSeq-inDrop (TruDrop) library struc- ture the P7 and P5 sides were swapped so that the se- quencing primer and flow cell binding site for the cell barcode + UMI side of the library followed Illumina’s TruSeq libraries. The transcript side of the library now uses the P7 structure of TruSeq [14, 15]. The TruDrop library preparation fol- lows the same steps as previously published for the V2 library with the substitution of the following primers for their V2 counterparts:.

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

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Preparation of NGS libraries with tagmented chromatins of various cells by using SALP. We performed SALP-seq with 100,000 GM12878, HeLa, HepG2, and 293T cells, and various numbers of HepG2 cells and 500). The gDNA was then purified from the mixture by using standard phe- nol:chloroform extraction as described [23]. Preparation of NGS libraries with Hind III-digested and sonicated DNA by using SALP.

A comparison of RNA extraction and sequencing protocols for detection of small RNAs in plasma

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highly represented miRNAs from each RNA extraction/library preparation combination. 10 miRNAs were higher by both library preparation methods in MagnaZol than miRNeasy and 2 miRNAs higher in miRNeasy than MagnaZol.

Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

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In our efforts to improve the technology, we have realized that purification of immunoprecipitated DNA and storage of the purified DNA, two steps that have received little attention, are critical for successful library preparation and overall ChIP-seq quality. The PCR amplification-based library preparation method is well accepted in ChIP-seq applications. There are some reports that less than 1 ng of DNA can be used for ChIP-seq library preparation [4].

Effects of sample age on data quality from targeted sequencing of museum specimens: What are we capturing in time?

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A salt solution was then added to the abdomen preparation (after the abdomen was removed and cleaned), as per the recommended protocol [62].. Library preparation. Library preparation was broadly based on the procedures outlined in [20], using the standard steps for NGS library preparation (i.e., end repair, adaptor ligation and fill-in, and indexing PCR), but with several modifications due to the fragmented nature of the starting material.

Reliable multiplex sequencing with rare index mis-assignment on DNB-based NGS platform

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Second, in the WGS-like library preparation method, balancing li- braries with indexes 41 – 48 were mixed into the pooled libraries (index 1 – 8). Unlike the mis-assignment of in- dexes 1 – 8, which includes all the contamination starting from library preparation, the mis-assignment of indexes 41 – 48 only represents the steps after DNB preparation..

High-throughput ChIPmentation: Freely scalable, single day ChIPseq data generation from very low cell-numbers

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ChIPmentation library preparation. DNA purification was carried out using Qiagen MinElute PCR Purification Kit. Fifteen microliters of PCR master mix and 5 μl of primer mix (Nextera, Illumina) was added to 20 μl of eluted DNA, and libraries were amplified as de- scribed for HT-ChIPmentation libraries.. Two nanograms of DNA was used for library preparation using the Thru- PLEX DNA-seq kit (Rubicon Genomics) with 11 cycles of PCR amplification..

A comparative analysis of RNA sequencing methods with ribosome RNA depletion for degraded and low-input total RNA from formalin-fixed and paraffin-embedded samples

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Comparison of four RNA library preparation kits for FFPE samples. The consistency of transcript quantification of four RNA library preparation kits with FFPE samples. Comparison of mapping data using HISAT and STAR in FF and FFPE samples. RNA-seq: RNA sequencing. Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples.