Unknown,Transcriptomics,Genomics,Proteomics

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MAQC-III/SEQC Main Study ABCD titration experiment, ERCC spike-in profiles and gene-level estimates for a selected test set

ABSTRACT: Comparison to RNA-Seq showed different strengths and weaknesses for different regimes of expression strength. At sufficient read-depth, both platforms seemed comparable for gene-level expression profiling. Comparison to RNA-Seq showed comparable accuracy and precision with microarrays for ERCC spike-ins at medium to high expression levels. At low expression levels, the array showed signal attenuation but better precision, while RNA-Seq maintained accuracy albeit with highly inflated variance. In summary, both platforms can be meaningfully applied in a similar range of expression strength. Assessment of (precision, accuracy, reproducibility, mutual information, titration order consistency, and known mixing ratio recovery) by measurement of known-ratio mixtures of two RNA reference samples. The array probes 776 complex genes representative of the AceView gene model annotation, as well as 92 ERCC spike-in controls.

ORGANISM(S): Homo sapiens

SUBMITTER: Pawel Labaj

PROVIDER: E-GEOD-48016 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:While the importance of random sequencing errors decreases at higher DNA or RNA sequencing depths, systematic sequencing errors (SSEs) dominate at high sequencing depths and can be difficult to distinguish from biological variants. These SSEs can cause base quality scores to underestimate the probability of error at certain genomic positions, resulting in false positive variant calls, particularly in mixtures such as samples with RNA editing, tumors, circulating tumor cells, bacteria, mitochondrial heteroplasmy, or pooled DNA. Most algorithms proposed for correction of SSEs require a training data set, which is typically either from a part of the data set being M-bM-^@M-^\recalibratedM-bM-^@M-^] (Genome Analysis ToolKit, or GATK) or from a separate data set with special characteristics (SysCall). Here, we combine the advantages of these approaches by adding synthetic RNA spike-in standards to human RNA, and use GATK to recalibrate base quality scores with reads mapped to the spike-in standards. Compared to conventional GATK recalibration that uses reads mapped to the genome, spike-ins improve the accuracy of Illumina base quality scores by a mean of 5 units, and by as much as 13 units M-BM- at CpG sites. In addition, since reads mapping to the genome are not used for recalibration, our method allows run-specific recalibration even for the many species without a comprehensive and accurate SNP database. We also use GATK with the spike-in standards to demonstrate that the Illumina RNA sequencing runs overestimate quality scores for AC, CC, GC, GG, and TC dinucleotides, while SOLiD has less dinucleotide SSEs but more SSEs for certain cycles. We conclude that using these DNA and RNA spike-in standards with GATK improves base quality score recalibration. Four human RNA samples with equimolar ERCC spike-in standards were sequenced on Illumina. Two human brain/liver/muscle RNA mixtures with dynamic range of ERCC spike-in standards were sequenced on SOLiD.

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MAQC-III/SEQC Main Study ABCD titration experiment, ERCC spike-in profiles and gene-level estimates for a selected test set

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