Project description:Case series of children and adolescents undergoing growth hormone stimulation testing for investigation of short stature. The aim of this study was to identify whether a machine learning approach utilising gene expression data could predict which short children would test positive for GHD and which would not.
Project description:We explored changes at gene-level or transcript-level in embryonic stem cells, before and after in vitro differentiation with retinoic acid. RNA was sequenced both via Illumina short reads, and with Oxford Nanopore Technology with cDNA and direct RNA sequencing.
Project description:New tools for improved long-read transcript assembly and coalescence with its short-read counterpart are required. Using our short- and long-read measurements from different cell lines with spiked-in standards, we systematically compared key parameters and biases in the read alignment and assembly of transcripts. We report a cDNA synthesis artifact in long-read datasets that impacts the identity and quantitation of assembled transcripts. We developed a computational pipeline to strand long-read cDNA libraries that markedly improves assembly of transcripts from long-reads. Incorporating stranded long-reads in a new hybrid assembly approach, we demonstrate its efficacy for improved characterization of challenging lncRNA transcripts. Our workflow can be applied to a wide range of transcriptomics datasets for superior demarcation of transcript ends and refined isoform structure, which can enable better differential gene expression analyses and molecular manipulations of transcripts.
Project description:New tools for improved long-read transcript assembly and coalescence with its short-read counterpart are required. Using our short- and long-read measurements from different cell lines with spiked-in standards, we systematically compared key parameters and biases in the read alignment and assembly of transcripts. We report a cDNA synthesis artifact in long-read datasets that impacts the identity and quantitation of assembled transcripts. We developed a computational pipeline to strand long-read cDNA libraries that markedly improves assembly of transcripts from long-reads. Incorporating stranded long-reads in a new hybrid assembly approach, we demonstrate its efficacy for improved characterization of challenging lncRNA transcripts. Our workflow can be applied to a wide range of transcriptomics datasets for superior demarcation of transcript ends and refined isoform structure, which can enable better differential gene expression analyses and molecular manipulations of transcripts.
Project description:We explored changes at gene-level or transcript-level in embryonic stem cells, before and after in vitro differentiation with retinoic acid. RNA was sequenced both via Illumina short reads, and with Oxford Nanopore Technology with cDNA and direct RNA sequencing.
Project description:We developed a software package STITCH (https://github.com/snijderlab/stitch) to perform template-based assembly of de novo peptide reads from antibody samples. As a test case we generated de novo peptide reads from protein G purified whole IgG from COVID-19 patients.
Project description:Background: Cell free DNA (cfDNA) in plasma has received increasing attention and has been studied in a broad range of clinical conditions implicating inflammation, cancer, and aging. However, few studies have focused on mitochondrial DNA (mtDNA) in the cell free form. This study characterized the size distribution and sequence characteristics of plasma cell free mtDNA (cf mtDNA) in humans.Methods and Results: We optimized DNA isolation and next-generation sequencing library preparation protocols to better retain short DNA fragments from plasma, and applied these optimized methods to plasma samples from patients with sepsis. After massive parallel sequencing, we verified that our methods can retain substantially shorter DNA fragments than the standard isolation method, resulting in an average of 11.5 fold increase in short DNA fragments yield (DNA < 100bp). We report that cf mtDNA in plasma is highly enriched in short-size cfDNA (30 ~ 60 bp), which is much shorter than the value previously reported (~140 bp). Motivated by this unique size distribution, we size-selected short cfDNA fragments from the sequencing library, which further increased the mtDNA recovery rate by an average of 10.4 fold. Using this approach we detected mixtures of different mtDNA sequences, termed heteroplasmy, in plasma from 3 patients. In one patient who previously received bone marrow transplantation, different minor allele frequencies were observed between plasma and white blood cells (WBC) at heteroplasmic mtDNA sites, consistent with mixed-tissue origin for plasma DNA.Conclusion: mtDNA in plasma exists as very short fragments that exhibit mtDNA heteroplasmy distribution differences from that found in a single organ/tissue. This study is the first report of genome wide identification of mtDNA heteroplasmy in human plasma. Our optimized method can be used to investigate the potential utility of cf mtDNA fragments and heteroplasmy as biomarkers in various diseases.