Amplification-free library preparation with SAFE Hi-C uses ligation products for deep sequencing to improve traditional Hi-C analysis

Abstract

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.

Systematic Chromatin Architecture Analysis inXenopus tropicalisReveals Conserved Three-Dimensional Folding Principles of Vertebrate Genomes

“…To find out if the biased pattern of DI and enrichment of CTCF and Rad21 are conserved through evolution, we carried out similar analysis using previously published data for human K562 11 and Drosophila S2 cells 45 . Strikingly, we found even more obvious patterns in human K562 for all the three parameters analyzed (Figure 3G, 3H, 3I and S3G, S3H, S3I) and even the five sub-clusters of cluster 2 showed obvious bias in DI, CTCF, and Rad21 binding (Figure 3J, 3K, and 3L).…”

Resolving a Systematic Error in STARR-seq for Quantitative Enhancer Activity Mapping

“…Higher resolution and bias-free Hi-C method like SAFE Hi-C 53 In sum, we present here a solution to solve a systematic flaw in STARR-seq, a method used widely now days for functional enhancer identification, and even being modified for silencer identification in mammalian genomes 54 . Resolving this systematic error is important for increasingly intensive efforts to identify all kinds of gene regulatory elements in the genomes of model organisms, and to understand how gene transcription is precisely controlled in both ubiquitous and cell type-specific ways during development and differentiation.…”

The role of mobile genetic elements in organic micropollutant degradation during biological wastewater treatment

“… The assembly of MGEs becomes easier adding physical DNA interactions information. PCR amplification produce biases by underrepresenting genomic short-distance ligations (overcame by SAFE Hi-C) High price ( Van Berkum et al., 2010 ; Burton et al., 2014 , Niu et al., 2019 ) Optical mapping The assembly of MGEs becomes easier by adding long-range information. Low resolution (1 kb approximately compared to the single base pair resolution of sequencing) No host cells can be identified.…”

“…Similar to other technologies such as short-read sequencing and single-cell sequencing, Hi-C harbors the bias of PCR amplification. However, this problem is currently overcome by SAFE Hi-C ( Niu et al., 2019 ). Finally, if the objective is to analyze extracellular DNA (exDNA), two recent methods have shown increased DNA quality and yields in wastewater samples.…”