Characterizing human full spectrum genetic variations is crucial for understanding human health and disease. The PacBio long-read HiFi sequencing platform,which provides longer read length, higher accuracy, and sequencing without PCR amplification, has significantly advanced genomic research in this field. One of the key advantages of PacBio HiFi data, compared to short-read data, is its enhanced sensitivity in detecting and characterizing large and complex structural variants (SVs). Utilizing PacBio HiFi long-read sequencing enables researchers to gain deeper insights into the mechanisms, evolutionary history, and disease associations of SVs, even at the level of fully reconstructed haplotypes¹. However, the PacBio platform (Sequel IIe/Revio) generally requires >5μg of high-molecular-weight (HMW) DNA, which can be challenging to obtain. This high input requirement may discourage researchers from working with human samples that are low in concentration or limited in quantity.

Novogene has deep expertise in the scientific service and extensive experience with the PacBio platform. Today, we are excited to officially launch our Low-Input Amplification-Free HiFi Genome Sequencing service (hereinafter referred to as “1μg low-input HiFi library”), designed specifically for human PacBio HiFi sequencing with low DNA input amounts (~1μg HMW DNA). Our optimized library preparation pipeline effectively addresses the challenges of constructing low DNA input libraries using the SMRTbell® prep kit 3.0, ensuring high data output and quality. Unlike other low-input library preparation methods, our approach avoids additional amplification steps, eliminating PCR-related issues such as base bias, PCR chimeras, and allele dropout². This optimization preserves the key advantages of standard PacBio long-read HiFi libraries. The single-cell data output can reach up to 100 G, with an average base quality value of up to Q38.

Testing Data of Novogene1μg low-input HiFi library 1. PacBio Revio HiFi Data from ~1μg Human Whole Blood Samples

Novogene selected whole blood samples ranging from 0.5 μg to 1.3 μg for testing on the PacBio Revio platform. The sequencing results are remarkable, achieving an average of 93.8 Gb HiFi data per Cell. Furthermore, the average N50 read length is approximately 15,842 bp, with a mean QV>30, highlighting the exceptional quality and reliability of Novogene 1μg low-input HiFi library. For a detailed overview, please refer to Table 1, where we present data for eight of the samples.

Table 1 PacBio Revio testing data of Novogene 1μg low-input HiFi library

2. The Analysis Results of Variation Identification using 1μg Low-Input HiFi library (1) Structural Variations (SVs)

Structural variations (SVs) are typically defined as the alterations in genomic structure that is larger than 50 bp, including insertions (INS), deletions (DEL), inversions (INV), duplications (DUP), and copy number variations (CNVs). SV detection has emerged as a critical area of human research, with PacBio HiFi long-read sequencing providing superior accuracy and comprehensiveness in this domain compared to short-read sequencing. To assess the performance of 1μg low-input HiFi library, we compare the SV detection results and its breakpoint (BND) information between standard input HiFi libraries and 1μg low-input HiFi library (Figure 1).

Figure 1 SV statistics results

INS: Insertion; DEL: Deletion; INV: Inversion; BND: Breakpoint; DUP: Duplication; CNV: Copy number variation; H: Low-input samples; S: Standard input HiFi sample.

(2) SNP/ InDel

In addition to large structural variations (SVs), the low-input library also demonstrates excellent performance in detecting SNPs and InDels (Table 2).

Table 2 SNP/ InDel statistics results

H: Low-input samples; S: Standard input HiFi sample.

The Novogene new 1μg low-input HiFi library enables successful library preparation with a low input of DNA (~1μg HMW DNA), while maintaining high-quality data output and base quality, achieving analysis levels comparable to standard libraries. With the launch of the new 1μg low-input HiFi library, we hope more researchers will explore the potential of PacBio long-read HiFi sequencing in the field of human research.

Reference

1. Ebert P, Audano PA, Zhu Q, et al. Haplotype-resolved diverse human genomes and integrated analysis of structural variation. Science. 2021 May 7;372(6537)
2. Hård J, Mold JE, Eisfeldt J, et al. Long-read whole-genome analysis of human single cells. Nat Commun. 2023 Aug 11;14(1):5164.