Project description:Chromatin immunoprecipitation analysis of CENH3 in the Arabidopsis thaliana accessions Col-0, Ler-0, Cvi-0 and Tanz-1 was performed in order to align reads to PacBio HiFi genome assemblies which contain complete centromere repeat arrays.
Project description:PacBio HiFi sequencing was performed on 48 barcoded patients' genomic DNA after a telobait-capture protocol to enrich for telomeric regions. The sequencing reads of each patient were de-multiplexed and presented as patient-specific PacBio CCS BAM files.
Project description:26 breast cancer patient serum EV derived RNA and 4 control serum EV derived RNA was sequenced to explore the diagnostic potential of EV. EV RNA was isolated by EXOQUICK® Exosome isolation and RNA purification kit for serum/plasma (System Biosciences,CA) and was reverse transcribed to make amplified cDNA by SMART seq v4 ultra-low input RNA kit (Takara Bio Inc, USA). A sequencing library was made using 1 ng of sheared cDNA using The Low Input Library Prep Kit v2 (Takara Bio Inc, USA). DNA unique Dual index kit was used to combine libraries for sequencing (Takara Bio Inc, USA). NOVASEQ 6000 was used for sequencing to generate 25 million paired end reads per sample.
Project description:Different Library Sample Preparation (LSP) allow the detection of a large common set of isoforms. However, each LSP also detects a smaller set of isoforms which are characterized both by lower coverage and lower FPKM than that observed for the common ones among LSPs. This characteristic is particularly critical in case of low input RNA NuGEN v2 LSP. The effect of statistical detection of alternative splicing considering low input LSP (NuGEN v2) with respect to high input LSP (TruSeq) was studied using a benchmark dataset, in which both synthetic reads and reads generated from high and low input LSPs were spiked-in. Statistical detection of alternative splicing was done using prototypes of bioinformatics analysis for isoform-reconstruction and exon-level analysis. Each available sample contains a total of 5 paired end replicates. 3 samples contain increasing numbers of spiked-in reads (20, 40, 80 millions) from NuGENv2 library preparation kit on a common TruSeq 1000ng background. 3 additional samples were built with the same approach, but spiked-in reads were collected from a TruSeq-based experiment. The remaining 6 samples follow the same approach of the previous 6, but the common background is based on a TruSeq library preparation on 100ng of material
Project description:Different Library Sample Preparation (LSP) allow the detection of a large common set of isoforms. However, each LSP also detects a smaller set of isoforms which are characterized both by lower coverage and lower FPKM than that observed for the common ones among LSPs. This characteristic is particularly critical in case of low input RNA NuGEN v2 LSP. The effect of statistical detection of alternative splicing considering low input LSP (NuGEN v2) with respect to high input LSP (TruSeq) was studied using a benchmark dataset, in which both synthetic reads and reads generated from high and low input LSPs were spiked-in. Statistical detection of alternative splicing was done using prototypes of bioinformatics analysis for isoform-reconstruction and exon-level analysis.
Project description:We used PacBio data to identify more reliable transcripts from hESC, based on which we can estimate gene/transcript abundance better from Illumina data. PacBio long reads and Illumina short reads were generated from the same hESC cell line H1. PacBio reads were error-corrected by Illumina reads to identify transcripts. rSeq is used to estimate gene/transcript abundance of the identified transcriptome.
Project description:BmN4 cells are cultured cells derived from Bombyx mori ovaries and widely used to study transposon silencing by PIWI-interacting RNAs (piRNAs). A high-accurate genome sequence of BmN4 cells is required to analyze the piRNA pathway using RNA-seq. The genome sequence of BmN4 cells was assembled using Pacific Biosciences (PacBio) HiFi and Oxford Nanopore technology Ultralong (ONT-UL) reads. Microscopic observation and image analysis showed that BmN4 cells were octoploid on average, and the number of chromosomes per cell was highly variable. We concluded the haplotype-resolved assembly of such a complex genome would be difficult; therefore, we assembled a consensus genome sequence. RNA-seq analysis of Siwi knockdown cells also revealed that Siwi-piRISC may target Countdown (Cd), an LTR retrotransposon. By comparing the consensus genome sequence with the reads, we identified differences between haplotypes, particulary structural variants, suggesting that some transposons, including Countdown, increased their copy number in BmN4 cells.