Establishment of reference standards for multifaceted mosaic variant analysis

Abstract

Detection of somatic mosaicism in non-proliferative cells is a new challenge in genome research, however, the accuracy of current detection strategies remains uncertain due to the lack of a ground truth. Herein, we sought to present a set of ultra-deep sequenced WES data based on reference standards generated by cell line mixtures, providing a total of 386,613 mosaic single-nucleotide variants (SNVs) and insertion-deletion mutations (INDELs) with variant allele frequencies (VAFs) ranging from 0.5% to 56%, as well as 35,113,417 non-variant and 19,936 germline variant sites as a negative control. The whole reference standard set mimics the cumulative aspect of mosaic variant acquisition such as in the early developmental stage owing to the progressive mixing of cell lines with established genotypes, ultimately unveiling 741 possible inter-sample relationships with respect to variant sharing and asymmetry in VAFs. We expect that our reference data will be essential for optimizing the current use of mosaic variant detection strategies and for developing algorithms to enable future improvements.

Comprehensive benchmarking and guidelines of mosaic variant calling strategies

“…1) produced a large set of robust mosaic variants, but it could also alter the composition of germline variant sites. We resolved the issue of loss of pure germline sites by preparing a separate set of reference standards that recovered germline sites 19 . Second, we noted that germline variants from multiple cell lines may be presented as somatic-like variants of low to medium VAF after mixing.…”

“…The benchmark dataset was prepared using a recently constructed reference standard 19 . Briefly, the reference standard material is a collection of 39 mixtures of six pregenotyped normal cell lines.…”

“…The integrity of the used cell lines was confirmed with short tandem repeats comparison and verified to have no mycoplasma contamination (Supplementary Notes). Diverse combinations and ratios of the mixtures generated 39 reference materials, harboring both abundant mosaic variants of VAF (Supplementary Table 1) and two types of negative control: nonvariant sites (Set A) and germline variants (Set B) 19 . The 39 reference materials were included in one of the three mixture categories (nine M1, 12 M2 and 18 M3) that represent the genotypes of distinct lineages.…”

“…The population frequency data was needed for default settings, dbSNP(b154) for Mosaic Hunter and panel of normal (1000g_pon.hg38.vcf.gz) from GATK resource bundle (https://console. cloud.google.com/storage/browser/genomics-public-data/resources/ broad/hg38/v0) for MT2, were applied after the removal of positive controls; this was done because the positive controls were attained from mutually exclusive germline variants 19 . Segmental duplication and simple repeats from UCSC were used to remove both the low-confident regions in the control sets and in the tools (MH, MF and DM) if used as a part of the algorithm 11,12,20 .…”

Early life vaccination reprograms the metabolism and function of myeloid cells in neonates

“…In brief, Trim Galore (version 0.6.5) [40] was used for adapter trimming. Clean reads were then aligned to mouse reference genome (mm10) with BWA (version 0.7.17) [41] and then filtered with default parameters using SAMtools (version 1.10) [42] and Picard (version 2.23.1) [43]. Narrow peaks were called using MACS2 (version 2.2.7.1) [44].…”