Biology subjects

Nanda, A. S.

Publications and source records attributed to Nanda, A. S..

3 recordsLinked to original sources

bioRxiv2022

Sensitive multimodal profiling of native DNA by transposase-mediated single-molecule sequencing

We present SMRT-Tag: a multiplexable, PCR-free approach for constructing low-input, single-molecule Pacific Biosciences (PacBio) sequencing libraries through Tn5 transposition. As proof-of-concept, we apply SMRT-Tag to resolve human genetic and epigenetic variation in gold-standard human reference samples. SMRT-Tag requires 1-5% as much input material as existing protocols (15,000 - 50,000 human cell equivalents) and enables highly-sensitive and simultaneous detection of single nucleotide variants, small insertions / deletions, and CpG methylation comparable to the current state-of-the-art. We further combine SMRT-Tag with in situ adenine methyltransferase footprinting of nuclei (SAMOSA-Tag) to facilitate joint analysis of nucleosome repeat length, CTCF occupancy, and CpG methylation on individual chromatin fibers in osteosarcoma cells. SMRT-Tag promises to enable basic and clinical research by offering scalable, sensitive, and multimodal single-molecule genomic and epigenomic analyses in rare cell populations.

genomics↗

bioRxiv2021

Single-fiber nucleosome density shapes the regulatory output of a mammalian chromatin remodeling enzyme

ATP-dependent chromatin remodelers regulate the DNA accessibility required of virtually all nuclear processes. Biochemical studies have provided insight into remodeler action at the nucleosome level, but how these findings translate to activity on chromatin fibers in vitro and in vivo remains poorly understood. Here, we present a massively multiplex single-molecule platform allowing high-resolution mapping of nucleosomes on fibers assembled on mammalian genomic sequences. We apply this method to distinguish between competing models for chromatin remodeling by the essential ISWI ATPase SNF2h: linker-length-dependent dynamic positioning versus fixed-linker-length static clamping. Our single-fiber data demonstrate that SNF2h operates as a density-dependent, length-sensing chromatin remodeler whose ability to decrease or increase DNA accessibility depends on single-fiber nucleosome density. In vivo, this activity manifests as different regulatory modes across epigenomic domains: at canonically-defined heterochromatin, SNF2h generates evenly-spaced nucleosome arrays of multiple nucleosome repeat lengths; at SNF2h-dependent accessible sites, SNF2h slides nucleosomes to increase accessibility of motifs for the essential transcription factor CTCF. Overall, our generalizable approach provides molecularly-precise views of the processes that shape nuclear physiology. Concurrently, our data illustrate how a mammalian chromatin remodeling enzyme can effectively sense nucleosome density to induce diametrically-opposed regulatory effects within the nucleus.

genomics↗

bioRxiv2020

Dissecting mutational mechanisms underpinning signatures caused by replication errors and endogenous DNA damage

Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. Here, we generate isogenic CRISPR-Cas9 knockouts ({Delta}) of 43 genes in human induced pluripotent stem cells, culture them in the absence of added DNA damage, and perform wholegenome sequencing of 173 daughter subclones. {Delta}OGG1, {Delta}UNG, {Delta}EXO1, {Delta}RNF168, {Delta}MLH1, {Delta}MSH2, {Delta}MSH6, {Delta}PMS1, and {Delta}PMS2 produce marked mutational signatures indicative of being critical mitigators of endogenous DNA changes. Detailed analyses reveal that 8-oxo-dG removal by different repair proteins is sequence-context-specific while uracil clearance is sequencecontext-independent. Signatures of mismatch repair (MMR) deficiency show components of C>A transversions due to oxidative damage, T>C and C>T transitions due to differential misincorporation by replicative polymerases, and T>A transversions for which we propose a reverse template slippage model. {Delta}MLH1, {Delta}MSH6, and {Delta}MSH2 signatures are similar to each other but distinct from {Delta}PMS2. We validate these gene-specificities in cells from patients with Constitutive Mismatch Repair Deficiency Syndrome. Based on these experimental insights, we develop a classifier, MMRDetect, for improved clinical detection of MMR-deficient tumors.

genomics↗