Project description:Gene expression profiles generated with RNA sequencing can be biased by RNA amount and methods utilised for cDNA library generation. Polymerase chain reaction (PCR) amplification can generate high PCR duplicate proportions and introduce bias in transcript counts. In this study, we investigate the impact of input amount and PCR cycle number on the PCR duplication rate and on the RNA-seq data quality. We used a range of inputs (1 ng - 1,000 ng) and assessed the PCR duplication rate using unique molecular identifiers (UMIs). For broader applicability, we sequenced the data on four different short-read sequencing platforms: Illumina NovaSeq 6000, Illumina NovaSeq X, Element Biosciences AVITI, and Singular Genomics G4. We highlight the limitations of using input amounts below 125 ng and the advantages for using UMIs for deduplication. We contrast the data obtained from different sequencers and discuss the benefits and drawbacks of using Illumina library conversion kits.
2024-03-13 | GSE261432 | GEO
Project description:"30X Illumina NovaSeq sequencing of 1000 Genomes project trios.”
Project description:Low-pass sequencing (sequencing a genome to an average depth less than 1× coverage) combined with genotype imputation has been proposed as an alternative to genotyping arrays for trait mapping and calculation of polygenic scores. To empirically assess the relative performance of these technologies for different applications, we performed low-pass sequencing (targeting coverage levels of 0.5× and 1×) and array genotyping (using the Illumina Global Screening Array (GSA)) on 120 DNA samples derived from African and European-ancestry individuals that are part of the 1000 Genomes Project. We then imputed both the sequencing data and the genotyping array data to the 1000 Genomes Phase 3 haplotype reference panel using a leave- one-out design. We evaluated overall imputation accuracy from these different assays as well as overall power for GWAS from imputed data, and computed polygenic risk scores for coronary artery disease and breast cancer using previously derived weights. We conclude that low-pass sequencing plus imputation, in addition to providing a substantial increase in statistical power for genome wide association studies, provides increased accuracy for polygenic risk prediction at effective coverages of ∼ 0.5× and higher compared to the Illumina GSA.
Project description:Purpose: The goals of this study are to compare the transcription changes of dermal adipocytes during hair cycling. Methods: Transcriptome of purified dermal adipocytes were generated by deep sequencing, using Illumina Novaseq 6000 sequencing system.
Project description:For the scRNA-seq analysis of human blood lymphocytes and NK cells, lymphocytes were sorted as CD45+ cells, and NK cells were sorted as CD3/CD19/CD20/CD14−CD7+ cells using FACS (SONY SH800S); the purity was above 95%. The cells from 4 elderly individuals were pooled in one tube and the cells from 4 young individuals were pooled in another tube. We then counted and resuspended the pooled cells at a concentration of 1000 cells/μL, aiming for an estimated 6000 cells per library, following the instructions of the single-cell 3ʹ solution v3 reagent kit (10X Genomics). Single-cell libraries were constructed strictly according to the manufacturer’s standard protocols. Each sequencing library was generated with a unique sample index. Libraries were sequenced on the Illumina NovaSeq 6000 system.
Project description:Purpose: The goals of this study are to compare the transcription difference of epididymal fat between Tfrcfl/fl and TfrcAKO mice after high fat diet treatment Methods: Transcriptome of epididymal adipose tissue was generated by deep sequencing, using Illumina Novaseq 6000 sequencing system.
Project description:This study compares gene expression in three strains (CPL2H1, CPL2H1 gtf1/gtf1 and CPL2H1 otf1/otf1) with the genetic background of Candida parapsilosis CLIB 214 (CBS 604). Total RNA was isolated using hot acid phenol extraction. A TruSeq stranded mRNA library was sequenced on an Illumina NovaSeq 6000 system.
