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Background
The tremendous output of massive parallel sequencing technologies requires automated robust and scalable sample preparation methods to fully exploit the new sequence capacity.Methodology
In this study, a method for automated library preparation of RNA prior to massively parallel sequencing is presented. The automated protocol uses precipitation onto carboxylic acid paramagnetic beads for purification and size selection of both RNA and DNA. The automated sample preparation was compared to the standard manual sample preparation.Conclusion/Significance
The automated procedure was used to generate libraries for gene expression profiling on the Illumina HiSeq 2000 platform with the capacity of 12 samples per preparation with a significantly improved throughput compared to the standard manual preparation. The data analysis shows consistent gene expression profiles in terms of sensitivity and quantification of gene expression between the two library preparation methods. 相似文献2.
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Jesper R. G?din Ferdinand M. van’t Hooft Per Eriksson Lasse Folkersen 《BMC bioinformatics》2015,16(1)
Background
One aspect in which RNA sequencing is more valuable than microarray-based methods is the ability to examine the allelic imbalance of the expression of a gene. This process is often a complex task that entails quality control, alignment, and the counting of reads over heterozygous single-nucleotide polymorphisms. Allelic imbalance analysis is subject to technical biases, due to differences in the sequences of the measured alleles. Flexible bioinformatics tools are needed to ease the workflow while retaining as much RNA sequencing information as possible throughout the analysis to detect and address the possible biases.Results
We present AllelicImblance, a software program that is designed to detect, manage, and visualize allelic imbalances comprehensively. The purpose of this software is to allow users to pose genetic questions in any RNA sequencing experiment quickly, enhancing the general utility of RNA sequencing. The visualization features can reveal notable, non-trivial allelic imbalance behavior over specific regions, such as exons.Conclusions
The software provides a complete framework to perform allelic imbalance analyses of aligned RNA sequencing data, from detection to visualization, within the robust and versatile management class, ASEset.Electronic supplementary material
The online version of this article (doi:10.1186/s12859-015-0620-2) contains supplementary material, which is available to authorized users. 相似文献4.
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Chandra Sekhar Reddy Chilamakuri Susanne Lorenz Mohammed-Amin Madoui Daniel Vodák Jinchang Sun Eivind Hovig Ola Myklebost Leonardo A Meza-Zepeda 《BMC genomics》2014,15(1)
Background
Recent developments in deep (next-generation) sequencing technologies are significantly impacting medical research. The global analysis of protein coding regions in genomes of interest by whole exome sequencing is a widely used application. Many technologies for exome capture are commercially available; here we compare the performance of four of them: NimbleGen’s SeqCap EZ v3.0, Agilent’s SureSelect v4.0, Illumina’s TruSeq Exome, and Illumina’s Nextera Exome, all applied to the same human tumor DNA sample.Results
Each capture technology was evaluated for its coverage of different exome databases, target coverage efficiency, GC bias, sensitivity in single nucleotide variant detection, sensitivity in small indel detection, and technical reproducibility. In general, all technologies performed well; however, our data demonstrated small, but consistent differences between the four capture technologies. Illumina technologies cover more bases in coding and untranslated regions. Furthermore, whereas most of the technologies provide reduced coverage in regions with low or high GC content, the Nextera technology tends to bias towards target regions with high GC content.Conclusions
We show key differences in performance between the four technologies. Our data should help researchers who are planning exome sequencing to select appropriate exome capture technology for their particular application.Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-449) contains supplementary material, which is available to authorized users. 相似文献6.
Erin N Smith Kristen Jepsen Mahdieh Khosroheidari Laura Z Rassenti Matteo D’Antonio Emanuela M Ghia Dennis A Carson Catriona HM Jamieson Thomas J Kipps Kelly A Frazer 《Genome biology》2014,15(7)
Accurate allele frequencies are important for measuring subclonal heterogeneity and clonal evolution. Deep-targeted sequencing data can contain PCR duplicates, inflating perceived read depth. Here we adapted the Illumina TruSeq Custom Amplicon kit to include single molecule tagging (SMT) and show that SMT-identified duplicates arise from PCR. We demonstrate that retention of PCR duplicate reads can imply clonal evolution when none exists, while their removal effectively controls the false positive rate. Additionally, PCR duplicates alter estimates of subclonal heterogeneity in tumor samples. Our method simplifies PCR duplicate identification and emphasizes their removal in studies of tumor heterogeneity and clonal evolution.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0420-4) contains supplementary material, which is available to authorized users. 相似文献7.
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Young Joo Jeon Yulin Zhou Yihan Li Qiwei Guo Jinchun Chen Shengmao Quan Ahong Zhang Hailing Zheng Xingqiang Zhu Jin Lin Huan Xu Ayang Wu Sin-Gi Park Byung Chul Kim Hee Jae Joo Hongliang Chen Jong Bhak 《PloS one》2014,9(10)
Objective
Recent non-invasive prenatal testing (NIPT) technologies are based on next-generation sequencing (NGS). NGS allows rapid and effective clinical diagnoses to be determined with two common sequencing systems: Illumina and Ion Torrent platforms. The majority of NIPT technology is associated with Illumina platform. We investigated whether fetal trisomy 18 and 21 were sensitively and specifically detectable by semiconductor sequencer: Ion Proton.Methods
From March 2012 to October 2013, we enrolled 155 pregnant women with fetuses who were diagnosed as high risk of fetal defects at Xiamen Maternal & Child Health Care Hospital (Xiamen, Fujian, China). Adapter-ligated DNA libraries were analyzed by the Ion Proton™ System (Life Technologies, Grand Island, NY, USA) with an average 0.3× sequencing coverage per nucleotide. Average total raw reads per sample was 6.5 million and mean rate of uniquely mapped reads was 59.0%. The results of this study were derived from BWA mapping. Z-score was used for fetal trisomy 18 and 21 detection.Results
Interactive dot diagrams showed the minimal z-score values to discriminate negative versus positive cases of fetal trisomy 18 and 21. For fetal trisomy 18, the minimal z-score value of 2.459 showed 100% positive predictive and negative predictive values. The minimal z-score of 2.566 was used to classify negative versus positive cases of fetal trisomy 21.Conclusion
These results provide the evidence that fetal trisomy 18 and 21 detection can be performed with semiconductor sequencer. Our data also suggest that a prospective study should be performed with a larger cohort of clinically diverse obstetrics patients. 相似文献15.
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