Low Input sequencing of Mtb

The implementation of Mycobacterium tuberculosis (Mtb) whole genome sequencing (WGS) in clinical samples is often limited by the small amounts of Mtb DNA available. Next-generation sequencing (NGS) typically requires high-quality DNA inputs of 10-1000ng and while techniques such as whole genome amplification (WGA) have shown promise in overcoming this limitation, they often introduce bias or require specialized equipment. This study aimed to evaluate specialized low-input DNA library preparation kits as alternatives to WGA and compare their performance with standard kits to determine whether they can produce comparable sequencing results without the need for amplification. We tested four library preparation kits: two specialized low-input kits (QIAseq Ultralow Input and xGen ssDNA Low-Input) and two standard kits (Illumina DNA Prep and Twist Library Preparation EF Kit), using Mycobacterium tuberculosis H37Rv DNA extracted from cultured samples. DNA was input at concentrations ranging from 10 ng to 0.001 ng, and the resulting libraries were sequenced on Illumina's MiniSeq platform. Sequencing performance was assessed using several metrics, including the percentage of reads passing filter (%PF), percentage of high-quality reads (Q30), normalized genome coverage, and duplication rates. The results indicated that the Twist and Illumina kits maintained stable sequencing quality across a wide range of DNA inputs, whereas the specialized kits (Qiagen and xGen) showed significant performance degradation at lower DNA inputs. Notably, the Illumina kit exhibited the best performance, with the lowest levels of adapter contamination and duplication rates. These findings suggest that standard enzymatic fragmentation-based library preparation kits, despite not being specifically designed for ultra-low input DNA, can outperform specialized low-input kits in sequencing GC-rich Mtb genomes at picogram levels without the need for WGA.