Biology subjects

Rzepka, M.

Publications and source records attributed to Rzepka, M..

3 recordsLinked to original sources

bioRxiv2024

Single-Molecule Barcoding Technology for Single-Cell Genomics

Recent advances in barcoding technologies have significantly enhanced the scalability of single-cell genomic experiments. However, large-scale experiments are still rare due to high costs, complex logistics, and laborintensive procedures. To facilitate the routine application of the largest scalability, it is critical to simplify the production and use of barcoding reagents. Here, we introduce AmpliDrop, a technology that initiates the barcoding process using a pool of inexpensive single-copy barcodes and integrates barcode multiplicity generation with tagging of cellular content into a single reaction driven by DNA polymerase during library preparation. The barcoding reactions are compartmentalized using an electronic pipette or a robotic or standalone liquid handling system. These innovations eliminate the need for barcoded beads and complex combinatorial indexing workflows and provide flexibility for a wide range of scales and tube formats, as well as compatibility with automation. We show that AmpliDrop is capable of capturing transcriptomes and chromatin accessibility, and it can also be adapted for user-customized applications, including antibody-based protein detection, bacterial or viral DNA detection, and CRISPR perturbations without dual guide RNA-expression vectors. We validated AmpliDrop by investigating the influence of short-term static culturing on cell composition in human forebrain organoids, revealing metabolic reprogramming in lineage progenitors.

molecular biology↗

bioRxiv2023

Targeted Phasing of 2-200 Kilobase DNA Fragments with a Short-Read Sequencer and a Single-Tube Linked-Read Library Method

In the human genome, heterozygous sites are genomic positions with different alleles inherited from each parent. On average, there is a heterozygous site every 1-2 kilobases (kb). Resolving whether two alleles in neighboring heterozygous positions are physically linked--that is, phased--is possible with a short-read sequencer if the sequencing library captures long-range information. TELL-Seq is a library preparation method based on millions of barcoded micro-sized beads that enables instrument-free phasing of a whole human genome in a single PCR tube. TELL-Seq incorporates a unique molecular identifier (barcode) to the short reads generated from the same high-molecular-weight (HMW) DNA fragment (known as linked-reads). However, genome-scale TELL-Seq is not cost-effective for applications focusing on a single locus or a few loci. Here, we present an optimized TELL-Seq protocol that enables the cost-effective phasing of enriched loci (targets) of varying sizes, purity levels, and heterozygosity. Targeted TELL-Seq maximizes linked-read efficiency and library yield while minimizing input requirements, fragment collisions on microbeads, and sequencing burden. To validate the targeted protocol, we phased seven 180-200 kb loci enriched by CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis, four 20 kb loci enriched by CRISPR/Cas9-mediated protection from exonuclease digestion, and six 2-13 kb loci amplified by PCR. The selected targets have clinical and research relevance (BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, and PKI3CA). These analyses reveal that targeted TELL-Seq provides a reliable way of phasing allelic variants within targets (2-200 kb in length) with the low cost and high accuracy of short-read sequencing.

genomics↗

bioRxiv2022

Bile acid-CoA:amino acid N-acyltransferase gene knockout alters early life development, the gut microbiome and reveals unusual bile acid conjugates in mice

Bile acids are steroid detergents in bile that contribute to fat absorption, cell signaling and microbiome interactions in mammals. The final step in their synthesis is amino acid conjugation with either glycine or taurine to a cholic acid or chenodeoxycholic acid backbone in the liver by the enzyme bile acid-CoA:amino acid N-acyltransferase (BAAT). Here, we describe the microbial, chemical, and physiological consequences of BAAT gene deletion in mice. BAAT-/- mice were underweight after weaning but quickly exhibited catch-up growth. At 3-weeks-of-age, KO animals had increased phospholipid excretion and decreased subcutaneous fat pad mass, glycogen staining in hepatocytes and vitamin A stores in the liver, but these phenotypes were less marked in adulthood. Their bile acid (BA) pool was highly altered throughout the 8-weeks of life but was not completely devoid of conjugated BAs. These animals had 27-fold lower amounts of taurine-conjugated BAs than wildtype in their liver, but similar concentrations of glycine-conjugated BAs and higher microbially-conjugated BAs. The BA pool in BAAT-/- was enriched in a variety of unusual bile acids that were putatively sourced from cysteamine conjugation with subsequent oxidation and methylation of the sulfur group to mimic taurine. KO mice also had an altered microbiome, but most strongly in the first 3-weeks, indicating bile acid conjugation is important for proper microbiome development during the postnatal period. Finally, antibiotic treatment increased taurine, glycine, and the unusually conjugated BAs in BAAT-/- animals, indicating the microbiome was not the likely source of the conjugation. Instead, BA conjugation in KO animals was likely derived from the peroxisomal acyltransferases ACNAT1 and ACNAT2, which are duplications of BAAT in the mouse genome, but inactivated in humans. This study demonstrates that BA conjugation is important for early life development in mice and is facilitated by other host or microbial enzymes besides BAAT in a manner that results in molecular mimics of taurine that may rescue pathological phenotypes.

microbiology↗