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PCR amplification of Hi-C libraries introduces unusable duplicates and results in a biased representation of chromatin interactions. We present a simplified, fast, and economically efficient Hi-C library preparation procedure, SAFE Hi-C, which generates sufficient non-amplified ligation products for deep sequencing from 30 million Drosophila cells. Comprehensive analysis of the resulting data shows that amplification-free Hi-C preserves higher complexity of chromatin interaction and lowers sequencing depth for the same number of unique paired reads. For human cells which have a large genome, SAFE Hi-C recovers enough ligated fragments for direct high-throughput sequencing without amplification from as few as 250,000 cells. Comparison with published in situ Hi-C data from millions of human cells demonstrates that amplification introduces distance-dependent amplification bias, which results in an increased background noise level against genomic distance. With amplification bias avoided, SAFE Hi-C may produce a chromatin interaction network more faithfully reflecting the real three-dimensional genomic architecture.

Viable pathogenic bacteria are major biohazards that pose a significant threat to food safety. Despite the recent developments in detection platforms, multiplex identification of viable pathogens in food remains a major challenge. A novel strategy is developed through direct metatranscriptome RNA-seq and multiplex RT-PCR amplicon sequencing on Nanopore MinION to achieve real-time multiplex identification of viable pathogens in food. Specifically, this study reports an optimized universal Nanopore sample extraction and library preparation protocol applicable to both Gram-positive and Gramnegative pathogenic bacteria, demonstrated using a cocktail culture of E. coli O157:H7, Salmonella enteritidis, and Listeria monocytogenes, which were selected based on their impact on economic loss or prevalence in recent outbreaks. Further evaluation and validation confirmed the accuracy of direct metatranscriptome RNA-seq and multiplex RT-PCR amplicon sequencing using Sanger sequencing and selective media. The study also included a comparison of different bioinformatic pipelines for metatranscriptomic and amplicon genomic analysis. MEGAN without rRNA mapping showed the highest accuracy of multiplex identification using the metatranscriptomic data. EPI2ME also demonstrated high accuracy using multiplex RT-PCR amplicon sequencing. In addition, a systemic comparison was drawn between Nanopore sequencing of the direct metatranscriptome RNA-seq and RT-PCR amplicons. Both methods are comparable in accuracy and time. Nanopore sequencing of RT-PCR amplicons has higher sensitivity, but Nanopore metatranscriptome sequencing excels in read length and dealing with complex microbiome and non-bacterial transcriptome backgrounds.

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