Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Impact of library preparation on downstream analysis and interpretation of RNA-seq data: comparison between Illumina PolyA and NuGEN Ovation protocol

ABSTRACT: Objectives: The sequencing by the PolyA selection is the most common approach for library preparation. With limited amount or degraded RNA, alternative protocols such as the NuGEN have been developed. However, it is not yet clear how the different library preparations affect the downstream analyses of the broad applications of RNA sequencing. Methods and Materials: Eight human mammary epithelial cell (HMEC) lines with high quality RNA were sequenced by Illumina’s mRNA-Seq PolyA selection and NuGEN ENCORE library preparation. The following analyses and comparisons were conducted: 1) the numbers of genes captured by each protocol; 2) the impact of protocols on differentially expressed gene detection between biological replicates; 3) expressed single nucleotide variant (SNV) detection; 4) non-coding RNAs, particularly lincRNA detection; and 5) intragenic gene expression. Results: Sequences from the NuGEN protocol had lower (75%) alignment rate than the PolyA (over 90%). The NuGEN protocol detected fewer genes (12-20% less) with a significant portion of reads mapped to non-coding regions. A large number of genes were differentially detected between the two protocols. About 17-20% of the differentially expressed genes between biological replicates were commonly detected between the two protocols. Significantly higher numbers of SNVs (5-6 times) were detected in the NuGEN samples, which were largely from intragenic and intergenic regions. The NuGEN captured fewer exons (25% less) and had higher base level coverage variance. While 6.3% of reads were mapped to intragenic regions in the PolyA samples, the percentages were much higher (20-25%) for the NuGEN samples. The NuGEN protocol did not detect more known non-coding RNAs such as lincRNAs, but targeted small and “novel” lincRNAs. Conclusion: Different library preparations can have significant impacts on downstream analysis and interpretation of RNA-seq data. The NuGEN provides an alternative for limited or degraded RNA but it has limitations for some RNA-seq applications. Eight human mammary epithelial cell (HMEC) lines with high quality RNA were sequenced by Illumina’s mRNA-Seq PolyA selection and NuGEN ENCORE library preparation. The goal was to look the impacts of protocols on results and intepretation.

ORGANISM(S): Homo sapiens

SUBMITTER: Zhifu Sun

PROVIDER: E-GEOD-47933 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Impact of library preparation on downstream analysis and interpretation of RNA-Seq data: comparison between Illumina PolyA and NuGEN Ovation protocol.

Sun Zhifu Z Asmann Yan W YW Nair Asha A Zhang Yuji Y Wang Liguo L Kalari Krishna R KR Bhagwate Aditya V AV Baker Tiffany R TR Carr Jennifer M JM Kocher Jean-Pierre A JP Perez Edith A EA Thompson E Aubrey EA

PloS one 20130819 8

<h4>Objectives</h4>The sequencing by the PolyA selection is the most common approach for library preparation. With limited amount or degraded RNA, alternative protocols such as the NuGEN have been developed. However, it is not yet clear how the different library preparations affect the downstream analyses of the broad applications of RNA sequencing.<h4>Methods and materials</h4>Eight human mammary epithelial cell (HMEC) lines with high quality RNA were sequenced by Illumina's mRNA-Seq PolyA sele ...[more]

PMID: 23977132

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Project description:Objectives: The sequencing by the PolyA selection is the most common approach for library preparation. With limited amount or degraded RNA, alternative protocols such as the NuGEN have been developed. However, it is not yet clear how the different library preparations affect the downstream analyses of the broad applications of RNA sequencing. Methods and Materials: Eight human mammary epithelial cell (HMEC) lines with high quality RNA were sequenced by Illumina’s mRNA-Seq PolyA selection and NuGEN ENCORE library preparation. The following analyses and comparisons were conducted: 1) the numbers of genes captured by each protocol; 2) the impact of protocols on differentially expressed gene detection between biological replicates; 3) expressed single nucleotide variant (SNV) detection; 4) non-coding RNAs, particularly lincRNA detection; and 5) intragenic gene expression. Results: Sequences from the NuGEN protocol had lower (75%) alignment rate than the PolyA (over 90%). The NuGEN protocol detected fewer genes (12-20% less) with a significant portion of reads mapped to non-coding regions. A large number of genes were differentially detected between the two protocols. About 17-20% of the differentially expressed genes between biological replicates were commonly detected between the two protocols. Significantly higher numbers of SNVs (5-6 times) were detected in the NuGEN samples, which were largely from intragenic and intergenic regions. The NuGEN captured fewer exons (25% less) and had higher base level coverage variance. While 6.3% of reads were mapped to intragenic regions in the PolyA samples, the percentages were much higher (20-25%) for the NuGEN samples. The NuGEN protocol did not detect more known non-coding RNAs such as lincRNAs, but targeted small and “novel” lincRNAs. Conclusion: Different library preparations can have significant impacts on downstream analysis and interpretation of RNA-seq data. The NuGEN provides an alternative for limited or degraded RNA but it has limitations for some RNA-seq applications.

Project description:Thousands of long intergenic noncoding RNAs (lincRNAs) are encoded by the mammalian genome, which were reported to have multiple biological functions as transcriptional activators acting in cis 1 or trans 2, transcriptional repressors 3,4 or miRNAs decoys 5,6. However, the function of most lincRNAs has not yet been identified in vivo. Here, we demonstrate a role for linc-MYH, a novel long intergenic noncoding RNA, in adult fast-type myofibre specialization. Skeletal myofibre fast and slow phenotypes are established through differential expression of numerous fibre-specific genes7. We show linc-MYH and the fast MYH genes share a common enhancer located in the fast MYH genes locus and regulated by the Six1 homeoproteins. Muscle-specific Six1 mutant mice show increased expression of slow-type genes, and downregulation of linc-MYH and fast-type genes. linc-MYH function revealed by in vivo knockdown and wide transcriptomic analysis, is in fine to prevent expression of genes ensuring slow muscle contractile properties, and to increase fast-type muscle gene expression in fast-type myofibres. Thus, formation of efficient fast sarcomeric units and appropriate Ca++ cycling and excitation/contraction/relaxation coupling in fast- myofibres is achieved through the coordiante control of fast MYHs and linc-MYH expression by a Six bound enhancer. Ten Î¼g of shRNA-expressing vector were introduced into TA muscles of 8 week-old mice by electroporation. Two weeks following electroporation, TA myofibres expressing GFP were dissected under a Nikon SMZ1500 stereo microscope and frozen in liquid nitrogen before processing. The efficiency of each shRNA was established by determination of linc-MYH transcript levels in TA muscles transfected by each shlincMYH. The shRNA against 5'- TTCTGCTCACCACCTACAATT-3' sequence was selected for the knockdown experiment. After validation of RNA quality with the Bioanalyzer 2100 (using Agilent RNA6000 nano chip kit), 50 ng of total RNA were reverse transcribed following the Ovation PicoSL WTA System (Nugen). Briefly, the resulting double-strand cDNA was used for amplification based on SPIA technology. After purification according to Nugen protocol, 5 Î¼g of single strand DNA was used for generation of Sens Target DNA using Ovation Exon Module kit (Nugen). 2.5 Î¼g of Sens Target DNA were fragmented and labelled with biotin using Encore Biotin Module kit (Nugen). After control of fragmentation using Bioanalyzer 2100, the cDNA was then hybridized to GeneChipÂ® Mouse Gene 1.0 ST (Affymetrix) at 45Â°C for 17 hours. After overnight hybridization, the ChIPs were washed using the fluidic station FS450 following specific protocols (Affymetrix) and scanned using the GCS3000 7G. The scanned images were then analyzed with Expression Console software (Affymetrix) to obtain raw data (cel files) and metrics for Quality Controls. The analysis of some of these metrics and the study of the distribution of raw data show no outlier experiment. Gastrocnemius muscles were collected from cSix1 KO and control mice. Total RNAs were extracted by Trizol Reagent (Invitrogen) according to manufacturer's instruction. After validation of RNA quality with the Bioanalyzer 2100 (using Agilent RNA6000 nano chip kit), 50 ng of total RNA were reverse transcribed following the Ovation PicoSL WTA System (Nugen). Briefly, the resulting double-strand cDNA was used for amplification based on SPIA technology. After purification according to Nugen protocol, 5 Î¼g of single strand DNA was used for generation of Sens Target DNA using Ovation Exon Module kit (Nugen). 2.5 Î¼g of Sens Target DNA were fragmented and labelled with biotin using Encore Biotin Module kit (Nugen). After control of fragmentation using Bioanalyzer 2100, the cDNA was then hybridized to GeneChipÂ® Mouse Gene 1.0 ST (Affymetrix) at 45Â°C for 17 hours. After overnight hybridization, the ChIPs were washed using the fluidic station FS450 following specific protocols (Affymetrix) and scanned using the GCS3000 7G. The scanned images were then analyzed with Expression Console software (Affymetrix) to obtain raw data (cel files) and metrics for Quality Controls. The analysis of some of these metrics and the study of the distribution of raw data show no outlier experiment.

Dataset Information

Impact of library preparation on downstream analysis and interpretation of RNA-seq data: comparison between Illumina PolyA and NuGEN Ovation protocol

Publications

Impact of library preparation on downstream analysis and interpretation of RNA-Seq data: comparison between Illumina PolyA and NuGEN Ovation protocol.

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