An assessment of a massively parallel sequencing approach for the identification of individuals from mass graves of the Spanish Civil War (1936-1939).

Next-generation sequencing technologies have opened new opportunities in forensic genetics. Here, we assess the applicability and performance of the MiSeq FGx™ & ForenSeq™ DNA Signature Prep Kit (Illumina) for the identification of individuals from the mass graves of the Spanish Civil War (1936-1939). The main limitations for individual identification are the low number of possible first-degree living relatives and the high levels of DNA degradation reported in previous studies. Massively parallel sequencing technologies enabling the analysis of hundreds of regions and prioritizing short length amplicons constitute a promising tool for this kind of approaches. In this study, we first explore the power of this new technology to detect first- and second-degree kinship given different scenarios of DNA degradation. Second, we specifically assess its performance in a set of low DNA input samples previously analyzed with CE technologies. We conclude that this methodology will allow identification of up to second-degree relatives, even in situations with low sequencing performance and important levels of allele drop-out; it is thus a technology that resolves previous drawbacks and that will allow a successful approximation to the identification of remains.

LABS: linear amplification-based bisulfite sequencing for ultrasensitive cancer detection from cell-free DNA.

Methylation-based liquid biopsies show promises in detecting cancer using circulating cell-free DNA; however, current limitations impede clinical application. Most assays necessitate substantial DNA inputs, posing challenges. Additionally, underrepresented tumor DNA fragments may go undetected during exponential amplification steps of traditional sequencing methods. Here, we report linear amplification-based bisulfite sequencing (LABS), enabling linear amplification of bisulfite-treated DNA fragments in a genome-wide, unbiased fashion, detecting cancer abnormalities with sub-nanogram inputs. Applying LABS to 100 patient samples revealed cancer-specific patterns, copy number alterations, and enhanced cancer detection accuracy by identifying tissue-of-origin and immune cell composition.

Population Genomics of Pooled Samples: Unveiling Symbiont Infrapopulation Diversity and Host-Symbiont Coevolution.

Microscopic symbionts represent crucial links in biological communities. However, they present technical challenges in high-throughput sequencing (HTS) studies due to their small size and minimal high-quality DNA yields, hindering our understanding of host-symbiont coevolution at microevolutionary and macroevolutionary scales. One approach to overcome those barriers is to pool multiple individuals from the same infrapopulation (i.e., individual host) and sequence them together (Pool-Seq), but individual-level information is then compromised. To simultaneously address both issues (i.e., minimal DNA yields and loss of individual-level information), we implemented a strategic Pool-Seq approach to assess variation in sequencing performance and categorize genetic diversity (single nucleotide polymorphisms (SNPs)) at both the individual-level and infrapopulation-level for microscopic feather mites. To do so, we collected feathers harboring mites (Proctophyllodidae: Amerodectes protonotaria) from four individual Prothonotary Warblers (Parulidae: Protonotaria citrea). From each of the four hosts (i.e., four mite infrapopulations), we conducted whole-genome sequencing on three extraction pools consisting of different numbers of mites (1 mite, 5 mites, and 20 mites). We found that samples containing pools of multiple mites had more sequencing reads map to the feather mite reference genome than did the samples containing only a single mite. Mite infrapopulations were primarily genetically structured by their associated individual hosts (not pool size) and the majority of SNPs were shared by all pools within an infrapopulation. Together, these results suggest that the patterns observed are driven by evolutionary processes occurring at the infrapopulation level and are not technical signals due to pool size. In total, despite the challenges presented by microscopic symbionts in HTS studies, this work highlights the value of both individual-level and infrapopulation-level sequencing toward our understanding of host-symbiont coevolution at multiple evolutionary scales.

Comparison of EM-seq and PBAT methylome library methods for low-input DNA.

DNA methylation is the most studied epigenetic mark involved in regulation of gene expression. For low input samples, a limited number of methods for quantifying DNA methylation genome-wide has been evaluated. Here, we compared a series of input DNA amounts (1-10ng) from two methylome library preparation protocols, enzymatic methyl-seq (EM-seq) and post-bisulfite adaptor tagging (PBAT) adapted from single-cell PBAT. EM-seq takes advantage of enzymatic activity while PBAT relies on conventional bisulfite conversion for detection of DNA methylation. We found that both methods accurately quantified DNA methylation genome-wide. They produced expected distribution patterns around genomic features, high C-T transition efficiency at non-CpG sites and high correlation between input amounts. However, EM-seq performed better in regard to library and sequencing quality, i.e. EM-seq produced larger insert sizes, higher alignment rates and higher library complexity with lower duplication rate compared to PBAT. Moreover, EM-seq demonstrated higher CpG coverage, better CpG site overlap and higher consistency between input series. In summary, our data suggests that EM-seq overall performed better than PBAT in whole-genome methylation quantification of low input samples.

Cell-free DNA methylome profiling by MBD-seq with ultra-low input.

Methylation signatures in cell-free DNA (cfDNA) have shown great sensitivity and specificity in the characterization of tumour status and classification of tumour types, as well as the response to therapy and recurrence. Currently, most cfDNA methylation studies are based on bisulphite conversion, especially targeted bisulphite sequencing, while enrichment-based methods such as cfMeDIP-seq are beginning to show potential. Here, we report an enrichment-based ultra-low input cfDNA methylation profiling method using methyl-CpG binding proteins capture, termed cfMBD-seq. We optimized the conditions for cfMBD capture by adjusting the amount of MethylCap protein along with using methylated filler DNA. Our data show high correlation between low input cfMBD-seq and standard MBD-seq (>1000 ng input). When compared to cfMEDIP-seq, cfMBD-seq demonstrates higher sequencing data quality with more sequenced reads passed filter and less duplicate rate. cfMBD-seq also outperforms cfMeDIP-seq in the enrichment of CpG islands. This new bisulphite-free ultra-low input methylation profiling technology has great potential in non-invasive and cost-effective cancer detection and classification.

Low-Input Whole-Genome Bisulfite Sequencing.

DNA methylation can regulate gene expression by modulating chromatin accessibility and transcription factor binding on promoter and enhancer regions. Whole-genome bisulfite sequencing (WGBS) represents the most informative and comprehensive analysis to profile the DNA methylation status of all the cytosines at single-base resolution. However, most of the available protocols recommend an amount of input DNA (50 ng-5µg) that makes the WGBS unsuitable for limited samples and cell populations. In this chapter, we provide complete protocol to perform WGBS libraries from very low-input DNA. This protocol is recommended for the analysis of the whole-genome DNA methylation pattern in rare cell populations, like a defined stem cell population isolated from animal models or human samples.

Two high-quality de novo genomes from single ethanol-preserved specimens of tiny metazoans (Collembola).

BACKGROUND: Genome sequencing of all known eukaryotes on Earth promises unprecedented advances in biological sciences and in biodiversity-related applied fields such as environmental management and natural product research. Advances in long-read DNA sequencing make it feasible to generate high-quality genomes for many non-genetic model species. However, long-read sequencing today relies on sizable quantities of high-quality, high molecular weight DNA, which is mostly obtained from fresh tissues. This is a challenge for biodiversity genomics of most metazoan species, which are tiny and need to be preserved immediately after collection. Here we present de novo genomes of 2 species of submillimeter Collembola. For each, we prepared the sequencing library from high molecular weight DNA extracted from a single specimen and using a novel ultra-low input protocol from Pacific Biosciences. This protocol requires a DNA input of only 5 ng, permitted by a whole-genome amplification step. RESULTS: The 2 assembled genomes have N50 values >5.5 and 8.5 Mb, respectively, and both contain ∼96% of BUSCO genes. Thus, they are highly contiguous and complete. The genomes are supported by an integrative taxonomy approach including placement in a genome-based phylogeny of Collembola and designation of a neotype for 1 of the species. Higher heterozygosity values are recorded in the more mobile species. Both species are devoid of the biosynthetic pathway for ß-lactam antibiotics known in several Collembola, confirming the tight correlation of antibiotic synthesis with the species way of life. CONCLUSIONS: It is now possible to generate high-quality genomes from single specimens of minute, field-preserved metazoans, exceeding the minimum contig N50 (1 Mb) required by the Earth BioGenome Project.

The USDA-ARS Ag100Pest Initiative: High-Quality Genome Assemblies for Agricultural Pest Arthropod Research.

The phylum Arthropoda includes species crucial for ecosystem stability, soil health, crop production, and others that present obstacles to crop and animal agriculture. The United States Department of Agriculture's Agricultural Research Service initiated the Ag100Pest Initiative to generate reference genome assemblies of arthropods that are (or may become) pests to agricultural production and global food security. We describe the project goals, process, status, and future. The first three years of the project were focused on species selection, specimen collection, and the construction of lab and bioinformatics pipelines for the efficient production of assemblies at scale. Contig-level assemblies of 47 species are presented, all of which were generated from single specimens. Lessons learned and optimizations leading to the current pipeline are discussed. The project name implies a target of 100 species, but the efficiencies gained during the project have supported an expansion of the original goal and a total of 158 species are currently in the pipeline. We anticipate that the processes described in the paper will help other arthropod research groups or other consortia considering genome assembly at scale.

Detection of mixed-strain infections by FACS and ultra-low input genome sequencing.

The epidemiological tracking of a bacterial outbreak may be jeopardized by the presence of multiple pathogenic strains in one patient. Nevertheless, this fact is not considered in most of the epidemiological studies and only one colony per patient is sequenced. On the other hand, the routine whole genome sequencing of many isolates from each patient would be costly and unnecessary, because the number of strains in a patient is never known a priori. In addition, the result would be biased by microbial culture conditions. Herein we propose an approach for detecting mixed-strain infection, providing C. difficile infection as an example. The cells of the target pathogenic species are collected from the bacterial suspension by the fluorescence activated cell sorting (FACS) and a shallow genome sequencing is performed. A modified sequencing library preparation protocol for low-input DNA samples can be used for low prevalence gut pathogens (< 0.1% of the total microbiome). This FACS-seq approach reduces diagnostics time (no culture is needed) and may promote discoveries of novel strains. Methodological details, possible issues and future directions for the sequencing of these natural pan-genomes are herein discussed.

A High-Quality De novo Genome Assembly from a Single Mosquito Using PacBio Sequencing.

A high-quality reference genome is a fundamental resource for functional genetics, comparative genomics, and population genomics, and is increasingly important for conservation biology. PacBio Single Molecule, Real-Time (SMRT) sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. Improvements in throughput and concomitant reductions in cost have made PacBio an attractive core technology for many large genome initiatives, however, relatively high DNA input requirements (~5 µg for standard library protocol) have placed PacBio out of reach for many projects on small organisms that have lower DNA content, or on projects with limited input DNA for other reasons. Here we present a high-quality de novo genome assembly from a single Anopheles coluzzii mosquito. A modified SMRTbell library construction protocol without DNA shearing and size selection was used to generate a SMRTbell library from just 100 ng of starting genomic DNA. The sample was run on the Sequel System with chemistry 3.0 and software v6.0, generating, on average, 25 Gb of sequence per SMRT Cell with 20 h movies, followed by diploid de novo genome assembly with FALCON-Unzip. The resulting curated assembly had high contiguity (contig N50 3.5 Mb) and completeness (more than 98% of conserved genes were present and full-length). In addition, this single-insect assembly now places 667 (>90%) of formerly unplaced genes into their appropriate chromosomal contexts in the AgamP4 PEST reference. We were also able to resolve maternal and paternal haplotypes for over 1/3 of the genome. By sequencing and assembling material from a single diploid individual, only two haplotypes were present, simplifying the assembly process compared to samples from multiple pooled individuals. The method presented here can be applied to samples with starting DNA amounts as low as 100 ng per 1 Gb genome size. This new low-input approach puts PacBio-based assemblies in reach for small highly heterozygous organisms that comprise much of the diversity of life.

Flow Cytometry and Direct Sequencing of Viruses.

In order to describe a novel uncultured viral species, it is essential to obtain the DNA sequence of their full genomes. The ability to distinguish the viral genome from the genome of its bacterial host is the major challenge of the modern viromics. The major obstacles for mining of viral genomes in metagenomic assemblies is bacterial contamination in viromes and low DNA input for sequencing.These obstacles can be overcome by flow cytometry that allows collecting free viral particles from environmental samples. In addition, fluorescence activated cell sorting reduces the bacterial contamination. By using optimized sequencing protocols, the ultra-low input DNA samples can be sequenced directly, without the need for whole genome amplification. This chapter provides details for staining of environmental viruses, flow cytometry, and direct sequencing of ultra-low input DNA samples on Illumina platform.

A MBD-seq protocol for large-scale methylome-wide studies with (very) low amounts of DNA.

We recently showed that, after optimization, our methyl-CpG binding domain sequencing (MBD-seq) application approximates the methylome-wide coverage obtained with whole-genome bisulfite sequencing (WGB-seq), but at a cost that enables adequately powered large-scale association studies. A prior drawback of MBD-seq is the relatively large amount of genomic DNA (ideally >1 µg) required to obtain high-quality data. Biomaterials are typically expensive to collect, provide a finite amount of DNA, and may simply not yield sufficient starting material. The ability to use low amounts of DNA will increase the breadth and number of studies that can be conducted. Therefore, we further optimized the enrichment step. With this low starting material protocol, MBD-seq performed equally well, or better, than the protocol requiring ample starting material (>1 µg). Using only 15 ng of DNA as input, there is minimal loss in data quality, achieving 93% of the coverage of WGB-seq (with standard amounts of input DNA) at similar false/positive rates. Furthermore, across a large number of genomic features, the MBD-seq methylation profiles closely tracked those observed for WGB-seq with even slightly larger effect sizes. This suggests that MBD-seq provides similar information about the methylome and classifies methylation status somewhat more accurately. Performance decreases with <15 ng DNA as starting material but, even with as little as 5 ng, MBD-seq still achieves 90% of the coverage of WGB-seq with comparable genome-wide methylation profiles. Thus, the proposed protocol is an attractive option for adequately powered and cost-effective methylome-wide investigations using (very) low amounts of DNA.

Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue.

Unlocking clinically translatable genomic information, including copy number alterations (CNA), from formalin-fixed paraffin-embedded (FFPE) tissue is challenging due to low yields and degraded DNA. We describe a robust, cost-effective low-coverage whole genome sequencing (LC WGS) method for CNA detection using 5 ng of FFPE-derived DNA. CN profiles using 100 ng or 5 ng input DNA were highly concordant and comparable with molecular inversion probe (MIP) array profiles. LC WGS improved CN profiles of samples that performed poorly using MIP arrays. Our technique enables identification of driver and prognostic CNAs in archival patient samples previously deemed unsuitable for genomic analysis due to DNA limitations.

Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics.

High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. Here we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diverse Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (∼100-1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics.