Synthetic construct
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA278810 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:High-throughput sequencing (HTS) has become a powerful tool for the detection of and sequence characterization of microRNAs (miRNA) and other small RNAs (sRNA). Unfortunately, the use of HTS data to determine the relative quantity of different miRNAs in a sample has been shown to be inconsistent with quantitative PCR and Northern Blot results. Several recent studies have concluded that the major contributor to this inconsistency is bias introduced during the construction of sRNA libraries for HTS and that the bias is primarily derived from the adaptor ligation steps; specifically where single stranded adaptors are sequentially ligated to the 3' and 5'-end of sRNAs using T4 RNA ligases. In this study we investigated the effects of ligation bias by using a pool of randomized ligation substrates, defined mixtures of miRNA sequences and several combinations of adaptors in HTS library construction. We show that like the 3' adaptor ligation step, the 5' adaptor ligation is also biased, not because of primary sequence, but instead due to secondary structures of the two ligation substrates. We find that multiple secondary structural factors influence final representation in HTS results. Our results provide insight about the nature of ligation bias and allowed us to design adaptors that reduce ligation bias and produce HTS results that more accurately reflect the actual concentrations of miRNAs in the defined starting material. 28 samples were sequenced and the libraries were made using various synthetic oligo mixtures and adaptor combinations
2015-05-15 | E-GEOD-67053 | biostudies-arrayexpress
Project description:High-throughput sequencing (HTS) has become a powerful tool for the detection of and sequence characterization of microRNAs (miRNA) and other small RNAs (sRNA). Unfortunately, the use of HTS data to determine the relative quantity of different miRNAs in a sample has been shown to be inconsistent with quantitative PCR and Northern Blot results. Several recent studies have concluded that the major contributor to this inconsistency is bias introduced during the construction of sRNA libraries for HTS and that the bias is primarily derived from the adaptor ligation steps; specifically where single stranded adaptors are sequentially ligated to the 3' and 5'-end of sRNAs using T4 RNA ligases. In this study we investigated the effects of ligation bias by using a pool of randomized ligation substrates, defined mixtures of miRNA sequences and several combinations of adaptors in HTS library construction. We show that like the 3' adaptor ligation step, the 5' adaptor ligation is also biased, not because of primary sequence, but instead due to secondary structures of the two ligation substrates. We find that multiple secondary structural factors influence final representation in HTS results. Our results provide insight about the nature of ligation bias and allowed us to design adaptors that reduce ligation bias and produce HTS results that more accurately reflect the actual concentrations of miRNAs in the defined starting material.
2015-05-15 | GSE67053 | GEO
Project description:The ligation step in RNA sequencing library generation is a known source of bias. We present the first comparison of the standard duplex adaptor protocol supplied by Life Technologies for use on the Ion Torrent PGM with an alternate single adaptor approach involving CircLigase (CircLig). We also investigate whether using the thermostable ligase Methanobacterium thermoautotrophicum RNA ligase K97A (Mth K97A) for the initial ligation step in the CircLigase protocol reduces bias. A pool of small RNA fragments of known composition was converted into a sequencing library using one of three protocols and sequenced on an Ion Torrent PGM. The single adaptor CircLigase-based approach significantly reduces, but does not eliminate, bias in Ion Torrent data. Using Mth K97A as part of the CircLig method does not further reduce bias.
2014-06-24 | E-MTAB-2566 | biostudies-arrayexpress
Project description:One method of directional cloning of fragmented mRNA is based on single strand RNA ligation, for which ligation bias occurrs if the adapters have single sequences. The sequencing bias affects the mRNA quantification and subsequently the differential expression analysis. High definition (HD) adapters [Sorefan et al 2012, Xu et al 2015] can be used to diminish the cloning bias during library construction. HD adapters and standard illumina adapters were used to construct mRNA-seq libraries for a side by side comparison.
2019-10-01 | GSE102510 | GEO
Project description:Cell free DNA (cfDNA) in human plasma carries abundant information on physiological condition, especially in cancer patients such as esophageal cancer. Next-generation sequencing (NGS) as a rapidly developed technology could decode the information effectively. As a key step of NGS, using existing methods to construct cfDNA sequencing libraries is limited by several shortcomings. In this study, we developed a new NGS library construction method for highly degraded DNA, cfDNA as example, based on Single strand Adaptor Library Preparation (SALP). With the high ligation efficiency of single strand adaptor (SSA) which overhangs 3 random bases at 3' end of a double-strand DNA, the new method could construct the sequencing library with high sensitivity without using specific enzymes except T4 DNA ligase and Taq polymerase. With the special designed barcode T adaptor (BTA), multiple libraries constructed from different samples can be amplified in unbiased strategy and facility to compare. Using this method, this study successfully sequenced and compared totally 20 cfDNA samples derived from esophageal cancer patients and healthy people in whole genome scale. This study also compared the chromatin state between cancer patients and healthy people using cfDNA, identified the significant difference between different health condition. Our findings extend the application of cfDNA beyond the analysis with degraded DNA fragments itself, to the transcription regulation level, which also provide an important clue for biopsy of esophageal cancer and other diseases.
2019-04-12 | GSE116345 | GEO
Project description:Modeling bias and variation in the stochastic processes of small RNA sequencing
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Project description:shRNA Sensor Analysis of 5' U converted shRNAs reveals 3' target bias exists in addition to 5' guide bias
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Project description:Oligonucleotide library Illumina Sequencing of Aptamers
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