Telomere-to-Telomere Assembly Improves Host Reads Removal in Metagenomic High-Throughput Sequencing of Human Samples

ABSTRACT Metagenomic high-throughput sequencing brings revolution to the study of human microbiome, clinical pathogen detection, discovery and infection diagnosis, but clinical samples often contain abundant human nucleic acids, leading to a high proportion of host reads. A high-quality human reference genome is essential for removing host reads to make downstream analyses faster and more accurate. The recently published complete human genome, Telomere-to-Telomere CHM13 assembly (T2T), though achieved great success immediately, has yet to be tested for metagenomic sequencing. In this study, we demonstrated the innovation that T2T brings to the field, using a diverse set of samples containing 4.97 billion reads sequenced from 165 libraries, on short- and long-read platforms. To exclude the effect of algorithms in comparison of the genomes, we benchmarked the per-read performance of state-of-the-art algorithms. For short reads, bwa mem was the best-performing algorithm, with positive median of differences (MD) and adjusted p-values <0.001 for all comparisons, while no consistent difference in overall performance was found for long reads algorithms. T2T, when compared to current reference genomes hg38 and YH, significantly improved the per-read sensitivity (MD: 0.1443 to 0.7238 percentage point, all adjusted p-values < 0.001) in removing host reads for all sequencers, and the per-read Mathew’s correlation coefficient (MCC) with T2T was also higher (MD: 1.063 to 16.41 percentage point, all adjusted p-values <0.001). Genomic location of reads exclusively mappable to T2T concentrated mainly in newly added regions. Misclassified reads generally resulted from low complexity sequences, contaminations in reference genomes and sequencing abnormalities. In downstream microbe detection procedures, T2T did not affect true positive calls but greatly reduced false positive calls. The improvement in the ability to correctly remove host reads foretells the success of T2T to serve as the next prevailing reference genome in metagenomic sequencing of samples containing human nucleic acids.