Optimized Workflow for Whole Genome and Transcriptome Next‐Generation Sequencing of Single Cells or Limited Nucleic Acid Samples

Abstract

Whole genome and whole transcriptome sequencing require orders of magnitude more of starting nucleic acid than what is found in single cells or other extremely limited samples. High fidelity amplification of this minute amount of nucleic acids is essential to overcome the limitations caused by the low input, degradation and contamination, and to ensure a sufficient amount of DNA for preparation of high complex and high quality next-generation sequencing (NGS) libraries. Recent technical advances in multiple displacement amplification (MDA) enable studies of rare cell types, heterogeneity of body fluids, tissues, environmental samples, and organisms that cannot be cultured. Several strategies for amplification of limiting amounts of nucleic acid have been described, with PCR being popular. However, PCR-based methods result in high error rates, lower library complexity, and lower coverage uniformity. In this article, a HiFi MDA is used to accurately amplify the limited material and to allow library preparation starting from high input, while reducing PCR cycling to achieve sufficient library yields. This article describes a complete workflow from cells and small quantities of DNA or RNA to NGS libraries for Illumina sequencing instruments. © 2023 QIAGEN GmbH. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Whole genome amplification from single cells Support Protocol 1: PicoGreen™ quantification of MDA amplified DNA Support Protocol 2: Purification of amplified DNA after MDA Basic Protocol 2: Whole transcriptome amplification from single cells Alternate Protocol: Whole transcriptome amplification from purified RNA Basic Protocol 3: Enrichment of complete small genomes using target-specific primers in MDA Basic Protocol 4: Complete viral RNA amplification using target-specific primers in MDA Basic Protocol 5: Enzymatic fragmentation and adapter ligation of MDA amplified material Basic Protocol 6: Normalization of library concentration using magnetic beads.