Project description:Next Generation Sequencing has proven to be an exceptionally powerful tool in the field of genomics and transcriptomics. With recent development it is nowadays possible to analyze ultra-low input sample material down to single cells. Nevertheless investigating such sample material still limits the analysis to either the genome or transcriptome, hence a combined analysis of both types of nucleic acids from the same sample material is still in demand.We developed a protocol which enables the combined analysis of the genome as well as the transcriptome by Next Generation Sequencing from ultra-low input samples. The protocol was evaluated by sequencing sub-colony structures from human embryonic stem cells containing 150 to 200 cells. The method can be adapted to any available sequencing system (e.g. Illumina, SOLiD, 454, etc.).To our knowledge, this is the first report where sub-colonies of human embryonic stem cells have been analyzed both at the genomic as well as transcriptome level. The method of this proof of concept study may find useful practical applications for cases where only a limited number of cells are available, e.g. for tissues samples from biopsies, circulating tumor cells and cells from early embryonic development. The results we present demonstrate that a combined analysis of genomic DNA and messenger RNA from ultra low input samples is feasible and can readily be applied to other cellular systems with limited material available.
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:CD34+ hematopoietic stem progenitor cells (HSPCs) from cryo-preserved blood or bone marrow were FACS sorted in TriZol and RNA was isolated according to the manufacturer’s protocol. SMARTer Ultra Low Input RNA kit for sequencing (Clontech, v4 Cat# 634891) was used to generate cDNA. Sequencing libraries were generated using TruSeq Nano DNA Sample Preparation kits (Illumina, Cat# 20015964), according to the low sample protocol and paired-end sequenced on a HiSeq 2500 or Novaseq 6000 (both Illumina).
Project description:Here, we report an enrichment-based ultra-low input cfDNA methylation profiling method using methyl-CpG binding proteins capture, termed cfMBD-seq. We optimized the conditions of cfMBD capture by adjusting the amount of MethylCap protein along with using methylated filler DNA. Our data showed that cfMBD-seq performs equally to the standard MBD-seq (>1000 ng input) even when using 1 ng DNA as the input. cfMBD-seq demonstrated equivalent sequencing data quality as well as similar methylation profile when compared to cfMeDIP-seq. We showed that cfMBD-seq outperforms cfMeDIP-seq in the enrichment of CpG islands. This new bisulfite-free ultra-low input methylation profiling technology has a great potential in non-invasive and cost-effective cancer detection and classification.
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.
| EGAD00001008626 | EGA
Project description:Ultra-low input DNA sequencing of natural pan-genomes of pathogens
