Novel commercial kits for whole genome library preparation for next-generation sequencing on Illumina platforms promise shorter workflows, lower inputs and cost savings. Time savings are achieved by ...employing enzymatic DNA fragmentation and by combining end-repair and tailing reactions. Fewer cleanup steps also allow greater DNA input flexibility (1 ng-1 μg), PCR-free options from 100 ng DNA, and lower price as compared to the well-established sonication and tagmentation-based DNA library preparation kits.
We compared the performance of four enzymatic fragmentation-based DNA library preparation kits (from New England Biolabs, Roche, Swift Biosciences and Quantabio) to a tagmentation-based kit (Illumina) using low input DNA amounts (10 ng) and PCR-free reactions with 100 ng DNA. With four technical replicates of each input amount and kit, we compared the kits' fragmentation sequence-bias as well as performance parameters such as sequence coverage and the clinically relevant detection of single nucleotide and indel variants. While all kits produced high quality sequence data and demonstrated similar performance, several enzymatic fragmentation methods produced library insert sizes which deviated from those intended. Libraries with longer insert lengths performed better in terms of coverage, SNV and indel detection. Lower performance of shorter-insert libraries could be explained by loss of sequence coverage to overlapping paired-end reads, exacerbated by the preferential sequencing of shorter fragments on Illumina sequencers. We also observed that libraries prepared with minimal or no PCR performed best with regard to indel detection.
The enzymatic fragmentation-based DNA library preparation kits from NEB, Roche, Swift and Quantabio are good alternatives to the tagmentation based Nextera DNA flex kit from Illumina, offering reproducible results using flexible DNA inputs, quick workflows and lower prices. Libraries with insert DNA fragments longer than the cumulative sum of both read lengths avoid read overlap, thus produce more informative data that leads to strongly improved genome coverage and consequently also increased sensitivity and precision of SNP and indel detection. In order to best utilize such enzymatic fragmentation reagents, researchers should be prepared to invest time to optimize fragmentation conditions for their particular samples.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Degradation-specific processes and variation in laboratory protocols can bias the DNA sequence composition from samples of ancient or historic origin. Here, we identify a novel artifact in sequences ...from historic samples of Atlantic cod (Gadus morhua), which forms interrupted palindromes consisting of reverse complementary sequence at the 5' and 3'-ends of sequencing reads. The palindromic sequences themselves have specific properties - the bases at the 5'-end align well to the reference genome, whereas extensive misalignments exists among the bases at the terminal 3'-end. The terminal 3' bases are artificial extensions likely caused by the occurrence of hairpin loops in single stranded DNA (ssDNA), which can be ligated and amplified in particular library creation protocols. We propose that such hairpin loops allow the inclusion of erroneous nucleotides, specifically at the 3'-end of DNA strands, with the 5'-end of the same strand providing the template. We also find these palindromes in previously published ancient DNA (aDNA) datasets, albeit at varying and substantially lower frequencies. This artifact can negatively affect the yield of endogenous DNA in these types of samples and introduces sequence bias.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The gene ankyrin-3 (ANK3) has been consistently associated with bipolar disorder (BD) in several genome-wide association studies (GWAS). The exact molecular mechanisms underlying this genetic ...association remain unknown. The discovery of a loss-of-function variant (rs41283526*G) in an alternatively spliced exon (ENSE00001786716) with a protective effect, suggested that elevated expression of this particular isoform could be a risk factor for developing the disorder. We developed a novel approach for measuring the expression level of all splice forms at a challenging genetic locus using a combination of droplet digital PCR and high-throughput sequencing of indexed PCR amplicons. The combined method was performed on a large collection of 568 postmortem brain samples of BD and SCZ cases and controls. We also studied the expression of the splice forms in a child-development cohort of 41 healthy males. We found that our approach can quantify the splice forms in brain samples, although with less precision than ddPCR. We detected highly significant differences in expression of splice forms and transcription start sites between brain regions, notably with higher expression of the BD-associated isoform in the corpus callosum compared to frontal tissue (mean fold change = 1.80, p < 1e-4). Although the patients in our sample expressed the BD-associated splice form at a similar level to controls, adolescents in our child-development cohort had a clearly higher expression level than younger children (mean fold change = 1.97, p = 5e-3). These results suggest that this ANK3 splice form may play a role in the myelin maturation of the human brain.
ChIP-seq is the current method of choice for genome-wide protein location analysis. Here, we present a native (non-cross-linked) ChIP procedure suitable for histone proteins, coupled with an ...efficient library preparation technique for subsequent next-generation sequencing. The method enables ChIP-seq starting with 50,000 or more cells.
Background
Activated T helper type 2 (Th2) cells are believed to play a pivotal role in allergic airway inflammation, but which cells attract and activate Th2 cells locally have not been fully ...determined. Recently, it was shown in an experimental human model of allergic rhinitis (AR) that activated monocytes rapidly accumulate in the nasal mucosa after local allergen challenge, where they promote recruitment of Th2 cells and eosinophils.
Objective
To investigate whether monocytes are recruited to the lungs in paediatric asthma.
Methods
Tissue samples obtained from children and adolescents with fatal asthma attack (n = 12), age‐matched non‐atopic controls (n = 9) and allergen‐challenged AR patients (n = 8) were subjected to in situ immunostaining.
Results
Monocytes, identified as CD68+S100A8/A9+ cells, were significantly increased in the lower airway mucosa and in the alveoli of fatal asthma patients compared with control individuals. Interestingly, cellular aggregates containing CD68+S100A8/A9+ monocytes obstructing the lumen of bronchioles were found in asthmatics (8 out of 12) but not in controls. Analysing tissue specimens from challenged AR patients, we confirmed that co‐staining with CD68 and S100A8/A9 was a valid method to identify recently recruited monocytes. We also showed that the vast majority of accumulating monocytes both in the lungs and in the nasal mucosa expressed matrix metalloproteinase 10, suggesting that this protein may be involved in their migration within the tissue.
Conclusions and clinical relevance
Monocytes accumulated in the lungs of children and adolescents with fatal asthma attack. This finding strongly suggests that monocytes are directly involved in the immunopathology of asthma and that these pro‐inflammatory cells are potential targets for therapy.
In the past decade, chromatin immunoprecipitation sequencing (ChIP-seq) has emerged as the dominant technique for those wishing to perform genome-wide protein:DNA profiling. Owing to the tissue- and ...cell-type-specific nature of epigenetic marks, the field has been driven towards obtaining data from ever-lower cell numbers. In this review, we focus on the methodological developments that have lowered input requirements and the biological findings they have enabled, as we strive towards the ultimate goal of robust single-cell ChIP-seq.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Linkage analysis and DNA sequencing in a family exhibiting an X-linked mental retardation (XLMR) syndrome, characterized by microcephaly, epilepsy, ataxia, and absent speech and resembling Angelman ...syndrome, identified a deletion in the SLC9A6 gene encoding the Na+/H+ exchanger NHE6. Subsequently, other mutations were found in a male with mental retardation (MR) who had been investigated for Angelman syndrome and in two XLMR families with epilepsy and ataxia, including the family designated as having Christianson syndrome. Therefore, mutations in SLC9A6 cause X-linked mental retardation. Additionally, males with findings suggestive of unexplained Angelman syndrome should be considered as potential candidates for SLC9A6 mutations.
Copper–silver ionization (CSI) is an in-house water disinfection method primarily installed to eradicate Legionella bacteria from drinking water distribution systems (DWDS). Its effect on the ...abundance of culturable Legionella and Legionella infections has been documented in several studies. However, the effect of CSI on other bacteria in DWDS is largely unknown. To investigate these effects, we characterized drinking water and biofilm communities in a hospital using CSI, in a neighboring building without CSI, and in treated drinking water at the local water treatment plant. We used 16S rDNA amplicon sequencing and Legionella culturing. The sequencing results revealed three distinct water groups: (1) cold-water samples (no CSI), (2) warm-water samples at the research institute (no CSI), and (3) warm-water samples at the hospital (after CSI; ANOSIM, p < 0.001). Differences between the biofilm communities exposed and not exposed to CSI were less clear (ANOSIM, p = 0.022). No Legionella were cultured, but limited numbers of Legionella sequences were recovered from all 25 water samples (0.2–1.4% relative abundance). The clustering pattern indicated local selection of Legionella types (Kruskal–Wallis, p < 0.001). Furthermore, one unclassified Betaproteobacteria OTU was highly enriched in CSI-treated warm water samples at the hospital (Kruskal–Wallis, p < 0.001).
For the compact Drosophila genome, several factors mediating insulator function, such as su(Hw) and dCTCF, have been identified. Recent analyses showed that both these insulator‐binding factors are ...functionally dependent on the same cofactor, CP190. Here we analysed genome‐wide binding of CP190 and dCTCF. CP190 binding was detected at CTCF, su(Hw) and GAF sites and unexpectedly at the transcriptional start sites of actively transcribed genes. Both insulator and transcription start site CP190‐binding elements are strictly marked by a depletion of histone H3 and, therefore, a loss of nucleosome occupancy. In addition, CP190/dCTCF double occupancy was seen at the borders of many H3K27me3 ‘islands’. As before, these sites were also depleted of H3. Loss of either dCTCF or CP190 causes an increase of H3 and H3K27 trimethylation at these sites. Thus, for both types of cis‐regulatory elements, domain borders and promoters, the chromatin structure is dependent on CP190.
Transcriptional enhancement of X-linked genes to compensate for the sex chromosome monosomy in Drosophila males is brought about by a ribonucleoprotein assembly called Male-Specific-Lethal or Dosage ...Compensation Complex (MSL-DCC). This machinery is formed in male flies and specifically associates with active genes on the X chromosome. After assembly at dedicated high-affinity "entry" sites (HAS) on the X chromosome, the complex distributes to the nearby active chromatin. High-resolution, genome-wide mapping of the MSL-DCC subunits by chromatin immunoprecipitation (ChIP) on oligonucleotide tiling arrays suggests a rather homogenous spreading of the intact complex onto transcribed chromatin. Coupling ChIP to deep sequencing (ChIP-seq) promises to map the chromosomal interactions of the DCC with improved resolution. We present ChIP-seq binding profiles for all complex subunits, including the first description of the RNA helicase MLE binding pattern. Exploiting the preferential representation of direct chromatin contacts upon high-energy shearing, we report a surprising functional and topological separation of MSL protein contacts at three classes of chromosomal binding sites. Furthermore, precise determination of DNA fragment lengths by paired-end ChIP-seq allows decrypting of the local complex architecture. Primary contacts of MSL-2 and MLE define HAS for the DCC. In contrast, association of the DCC with actively transcribed gene bodies is mediated by MSL-3 binding to nucleosomes. We identify robust MSL-1/MOF binding at a fraction of active promoters genome-wide. Correlation analyses suggest that this association reflects a function outside dosage compensation. Our comprehensive analysis provides a new level of information on different interaction modes of a multiprotein complex at distinct regions within the genome.
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