A reassessment of DNA-immunoprecipitation-based genomic profiling

Abstract

DNA immunoprecipitation followed by sequencing (DIP-seq) is a common enrichment method for profiling DNA modifications in mammalian genomes. However, the results of independent DIP-seq studies often show considerable variation between profiles of the same genome and between profiles obtained by alternative methods. Here we show that these differences are primarily due to the intrinsic affinity of IgG for short unmodified DNA repeats. This pervasive experimental error accounts for 50-99% of regions identified as 'enriched' for DNA modifications in DIP-seq data. Correction of this error profoundly altered DNA-modification profiles for numerous cell types, including mouse embryonic stem cells, and subsequently revealed novel associations among DNA modifications, chromatin modifications and biological processes. We conclude that both matched input and IgG controls are essential in order for the results of DIP-based assays to be interpreted correctly, and that complementary, non-antibody-based techniques should be used to validate DIP-based findings to avoid further misinterpretation of genome-wide profiling data.

No evidence for DNA N 6 -methyladenine in mammals

“…Next, we extended this analysis to 16 distinct 6mdA DIP-seq samples across four independent studies (6,7,9,19), revealing that most of the enriched peaks in all studies were located in repetitive elements overrepresented in both low complexity regions and simple repeats compared to other genomic fractions (52.7-and 27.7-fold, respectively) ( fig. S2, A and B), consistent with off-target binding (20). Although studies have suggested localization of 6mdA to long inter-spersed nuclear elements (LINEs) in mammals (7,8,21), our analysis showed highly inconsistent enrichment of 6mdA DIP-seq over LINEs between samples and studies (−8.9-to 3.6-fold over genomic) ( fig.…”

“…Artifactual mapping of 6mdA by DNA immunoprecipitation sequencing 6mdA antibodies are also used in DNA immunoprecipitation sequencing (DIP-seq) to generate genome-wide profiles of 6mdA (8)(9)(10)19). We recently reported major technical errors related to off-target binding of antibodies to DNA repeats in DIP-seq resulting in almost exclusively false-positive signals for 6mdA in vertebrate species Xenopus laevis, Danio rerio, and Mus musculus (20). Comparing published human 6mdA DIP-seq data (7) for paired genomic and WGA DNA revealed highly similar profiles with over 70% of natively enriched 6mdA peaks also found in the WGA sample ( Fig.…”

“…Immunodot blot was performed as previously described by us (15,20) with slight modification of antibody concentrations. Briefly, DNA was denatured at 95°C for 15 min in 400 mM NaOH and 10 mM EDTA at a volume of 200 l, followed by immediate cooling on wet ice and brief centrifugation.…”

Minimally invasive classification of pediatric solid tumors using reduced representation bisulfite sequencing of cell-free DNA: a proof-of-principle study

“…More recently, cfMeDIP-seq 37 was used to detect and classify adult cancers in an early stage. However, as this method is based on immunoprecipitation it has its own drawbacks 38 . These techniques all cover different parts of the methylome, and the optimal technique for sensitive tumor detection remains yet to be demonstrated.…”

What to do about those immunoprecipitation blues

“…According to the team, between 50% and 99% of enriched regions in DIP-seq data might be false positives. They found nonspecific enrichment of areas of the genome called short tandem repeats (STRs) 4 . Other known sources of bias include the fact that the 5-methylcytosine (5mC) antibody labs mainly use preferentially enriches genomic regions of low CG content and the most frequently used 5-hydroxymethylcytosine (5hmC) antibody does the same at highly modified regions of the genome.…”