illumina library preparation polya selection  (Azenta)

Journal: PLoS ONE

Article Title: Impact of Library Preparation on Downstream Analysis and Interpretation of RNA-Seq Data: Comparison between Illumina PolyA and NuGEN Ovation Protocol

doi: 10.1371/journal.pone.0071745

Figure Lengend Snippet: Y axis on the left for solid lines and Y axis on the right for dashed lines, representing millions of reads and numbers of genes captured, respectively. The samples from PolyA preparation are on the left side of the graph (with “.PolyA” extension on sample name) and the NuGEN samples are on the right (with “.ng” extension). Although the NuGEN samples had much higher sequence depth, the numbers of reads mapped to exon/gene were quite similar to the PolyA samples or slightly. The numbers of captured genes were lower in the NuGEN samples.

Article Snippet: Using pooled bronchial airway epithelial cell brushings, Beane et al. conducted RNA-seq for the libraries prepared by both the Illumina PolyA selection and the NuGEN Ovation System and demonstrated fairly good agreement for the common genes detected by both libraries (Pearson correlation r 0.59) .

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Impact of Library Preparation on Downstream Analysis and Interpretation of RNA-Seq Data: Comparison between Illumina PolyA and NuGEN Ovation Protocol

doi: 10.1371/journal.pone.0071745

Figure Lengend Snippet: A: Boxplot of RPKM normalized expression for 8 samples from the PolyA and 8 samples from the NuGEN library preparation (from left to right in the same order). Samples with the same color were the same RNA extract but prepared by either the PolyA or NuGEN protocol. B: The example scatter plot of RPKM expression of the same sample (sample BSO19B) between two library preparations. R - Pearson correlation coefficient. Red line – diagonal line for perfect correlation. Green line – fitted lowess cure for the data. C: Gene counts in different expression level binned by standardized expression RPKM. There were 20,156 genes detected in at least one sample and all these genes were used for the binning. Y-axis: number of genes detected in each expression range in RPKM. Samples on the left side are the PolyA and on the right are the NuGEN. NuGEN samples had more genes missed (in 0–1 range) than PolyA samples. Two genes are highly expressed (>10,000 rpkm) in the NuGEN samples but no genes were in this range in the PolyA samples. The ranges with * had more genes in the NuGEN samples than the PolyA samples.

Article Snippet: Using pooled bronchial airway epithelial cell brushings, Beane et al. conducted RNA-seq for the libraries prepared by both the Illumina PolyA selection and the NuGEN Ovation System and demonstrated fairly good agreement for the common genes detected by both libraries (Pearson correlation r 0.59) .

Techniques: Expressing

Journal: PLoS ONE

Article Title: Impact of Library Preparation on Downstream Analysis and Interpretation of RNA-Seq Data: Comparison between Illumina PolyA and NuGEN Ovation Protocol

doi: 10.1371/journal.pone.0071745

Figure Lengend Snippet: A: Differentially expressed genes at FDR 0.05 (highlighted in red) between non-involuted and involuted epithelial phenotypes of the PolyA samples by edgeR. X-axis is the average expression across groups (normalized at count per million) in log2 scale. The Y-axis is the log2 fold change between non-involuted and involuted samples. B: Differentially expressed genes at FDR 0.05 (highlighted in red) between non-involuted and involuted epithelial phenotypes of the NuGEN samples by edgeR. X-axis is the average expression across groups (normalized at count per million) in log2 scale. The Y-axis is the log2 fold change between non-involuted and involuted samples. C: Venn diagram for common and unique DEGs (differentially expressed genes) by the PolyA and the NuGEN preparations at FDR 0.05. D: The scatter plot of log2 fold changes for the 64 common genes in 3C. All genes are in the same direction and most genes agree very well in fold change estimate except some genes have higher fold changes in the NuGEN samples. E: Venn diagram for common and unique DEGs (differentially expressed genes) by the PolyA and the NuGEN preparations at raw p value <0.01. More genes are significant and overlapping but the common genes are in the similar proportion as with FDR cut-off (21% vs 17% of total DEGs by either). F: Comparison of 5 genes between RT-PCR, DEGs by the PolyA preparation, and DEGs by the NuGEN preparations. All genes are differentially expressed by three methods using un-adjusted p value. The fold change estimates agree better between the PolyA preparation and the RT-PCR than between the NuGEN preparation and RT-PCR.

Article Snippet: Using pooled bronchial airway epithelial cell brushings, Beane et al. conducted RNA-seq for the libraries prepared by both the Illumina PolyA selection and the NuGEN Ovation System and demonstrated fairly good agreement for the common genes detected by both libraries (Pearson correlation r 0.59) .

Techniques: Expressing, Comparison, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Impact of Library Preparation on Downstream Analysis and Interpretation of RNA-Seq Data: Comparison between Illumina PolyA and NuGEN Ovation Protocol

doi: 10.1371/journal.pone.0071745

Figure Lengend Snippet: A: The total number of SNVs by the NuGEN (solid lines) and the PolyA (dashed lines) preparation (all SNVs regardless of genomic positions). There are dramatic differences between the two library preparations. However, the differences between two callers, SNVmix and UnifiedGenotyper, are small. X-axis –samples. Y-axis – number of SNVs detected in millions. B. Detected SNV comparison between the PolyA and the NuGEN preparations. The common SNVs are low. C. The number of SNVs by genomic positions for each sample by the PolyA (left) and the NuGEN (right). Higher numbers of SNVs were detected in the NuGEN samples than the PolyA samples, particularly in the intragenic and intergenic regions. D. SNVs in the coding regions by functional impacts in the PolyA and NuGEN samples. Although the total SNVs in the coding region are similar, SNVs from the PolyA have more synonymous SNVs than non-synonymous SNVs yet these are reversed for the NuGEN samples.

Article Snippet: Using pooled bronchial airway epithelial cell brushings, Beane et al. conducted RNA-seq for the libraries prepared by both the Illumina PolyA selection and the NuGEN Ovation System and demonstrated fairly good agreement for the common genes detected by both libraries (Pearson correlation r 0.59) .

Techniques: Comparison, Functional Assay

Journal: PLoS ONE

Article Title: Impact of Library Preparation on Downstream Analysis and Interpretation of RNA-Seq Data: Comparison between Illumina PolyA and NuGEN Ovation Protocol

doi: 10.1371/journal.pone.0071745

Figure Lengend Snippet: A: Captured exons by either the PolyA or the NuGEN with the common and unique ones shown in the Venn diagram. B: For commonly captured exons (111,255), the PolyA have higher and more even coverage than the NuGEN sample. C: Compared to the PolyA library, the average exon coverage for the NuGEN is more spread, with many exons at very low coverage (higher density on the left) while a few with very high coverage (long tail on the right side). Although the low variance at the low covered exons, the variance for highly covered exons are larger than the PolyA library. D: The coefficient of variance in majority of exons is higher in the NuGEN preparation than the PolyA preparation. Blue line – diagonal line. E: An example exon coverage for gene RCAN1. The upper panel is from the PolyA library preparation and the lower is from the NuGEN preparation. Uneven coverage is more obvious in the NuGEN than in the PolyA. A exon skip in the NuGEN sample is also seen. Noted also is there are quite a few reads mapped into the intronic regions in the NuGEN sample. F: 3′ bias at transcript level, both in the PolyA and the NuGEN preparation. The NuGEN appears more obvious. The data is normalized by RPKM at exon level and the transcript level is standardized at percentile. G: The aggregated expression (coverage) from the NuGEN is lower than the PolyA.

Article Snippet: Using pooled bronchial airway epithelial cell brushings, Beane et al. conducted RNA-seq for the libraries prepared by both the Illumina PolyA selection and the NuGEN Ovation System and demonstrated fairly good agreement for the common genes detected by both libraries (Pearson correlation r 0.59) .

Techniques: Expressing

Journal: PLoS ONE

Article Title: Impact of Library Preparation on Downstream Analysis and Interpretation of RNA-Seq Data: Comparison between Illumina PolyA and NuGEN Ovation Protocol

doi: 10.1371/journal.pone.0071745

Figure Lengend Snippet: A: The percentage of mapped reads to intragenic regions are much higher in the NuGEN than the PolyA samples (left axis for the blue line), however the number of genes with detected intragenic expression is lower than or similar to the PolyA samples. B: intragenic expression coefficient of variance between the PolyA and the NuGEN samples. The NuGEN samples have much higher variance than the PolyA samples.

Article Snippet: Using pooled bronchial airway epithelial cell brushings, Beane et al. conducted RNA-seq for the libraries prepared by both the Illumina PolyA selection and the NuGEN Ovation System and demonstrated fairly good agreement for the common genes detected by both libraries (Pearson correlation r 0.59) .

Techniques: Expressing

Journal: PLoS ONE

Article Title: Impact of Library Preparation on Downstream Analysis and Interpretation of RNA-Seq Data: Comparison between Illumina PolyA and NuGEN Ovation Protocol

doi: 10.1371/journal.pone.0071745

Figure Lengend Snippet: Samples with “.pa” extension were from the PolyA preparation and the samples with “.ng” were from the NuGEN preparation. Y-axis – number of genes with at least 1 read mapped. Gene categories with * in the legend have higher numbers in the PolyA samples and those without are higher in the NuGEN samples.

Article Snippet: Using pooled bronchial airway epithelial cell brushings, Beane et al. conducted RNA-seq for the libraries prepared by both the Illumina PolyA selection and the NuGEN Ovation System and demonstrated fairly good agreement for the common genes detected by both libraries (Pearson correlation r 0.59) .

Techniques: