Large scale library generation for high throughput sequencing.

PLoS One

Science for Life Laboratory, Division of Gene Technology, Royal Institute of Technology (KTH), School of Biotechnology, Solna, Sweden.

Published: April 2011

Background: Large efforts have recently been made to automate the sample preparation protocols for massively parallel sequencing in order to match the increasing instrument throughput. Still, the size selection through agarose gel electrophoresis separation is a labor-intensive bottleneck of these protocols.

Methodology/principal Findings: In this study a method for automatic library preparation and size selection on a liquid handling robot is presented. The method utilizes selective precipitation of certain sizes of DNA molecules on to paramagnetic beads for cleanup and selection after standard enzymatic reactions.

Conclusions/significance: The method is used to generate libraries for de novo and re-sequencing on the Illumina HiSeq 2000 instrument with a throughput of 12 samples per instrument in approximately 4 hours. The resulting output data show quality scores and pass filter rates comparable to manually prepared samples. The sample size distribution can be adjusted for each application, and are suitable for all high throughput DNA processing protocols seeking to control size intervals.

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083417PMC
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019119PLOS

Publication Analysis

Top Keywords

high throughput
8
instrument throughput
8
size selection
8
large scale
4
scale library
4
library generation
4
generation high
4
throughput
4
throughput sequencing
4
sequencing background
4

Similar Publications

Breast cancer is a leading cause of cancer-related deaths among women globally. It is imperative to explore novel biomarkers to predict breast cancer treatment response as well as progression. Here, we collected six breast cancer samples and paired normal tissues for high-throughput sequencing.

View Article and Find Full Text PDF

A stable combination of non-stable genes outperforms standard reference genes for RT-qPCR data normalization.

Sci Rep

December 2024

Laboratoire de Recherche en Sciences Végétales, Equipe Génomique et Biotechnologie des Fruits, UMR 5546, CNRS, UPS, Toulouse INP, Université de Toulouse, Toulouse, France.

Gene expression profiling is of key importance in all domains of life sciences, as medicine, environment, and plants, for both basic and applied research. Despite the emergence of microarrays and high-throughput sequencing, qPCR remains a standard method for gene expression analyses, with its data normalization step being crucial for ensuring accuracy. Currently, the most widely used normalization method is based on the use of reference genes, assumed to be stably expressed across all experimental conditions.

View Article and Find Full Text PDF

The gut microbiota alterations interact with the pathogenesis and progression of chronic kidney disease (CKD). Probiotics have received wide attention as a potential management in CKD. We investigated the effects of Lactobacillus paracasei N1115 (LP N1115) on intestinal microbiota and related short-chain fatty acids (SCFAs) in end stage kidney disease patients on peritoneal dialysis (PD) in a single-center, prospective, randomized, double-blind, placebo-controlled study.

View Article and Find Full Text PDF

Metagenomic next-generation sequencing and galactomannan testing for the diagnosis of invasive pulmonary aspergillosis.

Sci Rep

December 2024

Department of Respiratory and Critical Care Medicine, Zhengzhou University People's Hospital, Henan Provincial People's Hospital, Weiwu Road No. 7, Zhengzhou, 450003, Henan, China.

To evaluate the diagnostic value of metagenomic next-generation sequencing (mNGS) and galactomannan (GM) testing in invasive pulmonary aspergillosis (IPA) and to compare mNGS with other diagnostic approaches (serum/bronchoalveolar lavage fluid (BALF)-GM and conventional microbiological tests (CMTs) including sputum smears and culture, BALF fungal culture, and bronchial brushing). In all, 237 patients were enrolled in this retrospective study, including 120 patients with IPA and 117 with non-IPA pulmonary infections treated at Henan Provincial People's Hospital between June 2021 and February 2024. The diagnostic performance of mNGS was compared to conventional diagnostic methods including serum GM, BALF-GM, sputum smear microscopy, sputum culture, bronchial brushings, and BALF culture.

View Article and Find Full Text PDF

Genome-wide analysis of alternative splicing differences in hepatic ischemia reperfusion injury.

Sci Rep

December 2024

Department of Minimally Invasive Hepatic Surgery, Key Laboratory of Hepatosplenic Surgery, the First Affiliated Hospital of Harbin Medical University, Ministry of Education, Harbin, Heilongjiang, China.

Alternative splicing (AS) contributes to transcript and protein diversity, affecting their structure and function. However, the specific transcriptional regulatory mechanisms underlying AS in the context of hepatic ischemia reperfusion (IR) injury in mice have not been extensively characterized. In this study, we investigated differentially alternatively spliced (DAS) genes and differentially expressed transcripts (DETs) in a mouse model of hepatic IR injury using the high throughput RNA sequencing (RNA-seq) analysis and replicate multivariate analysis of transcript splicing (rMATS) analysis.

View Article and Find Full Text PDF

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!