Semi-Automated Library Preparation for High-Throughput DNA Sequencing Platforms

Next-generation sequencing platforms are powerful technologies, providing gigabases of genetic information in a single run. An important prerequisite for high-throughput DNA sequencing is the development of robust and cost-effective preprocessing protocols for DNA sample library construction. Here we report the development of a semi-automated sample preparation protocol to produce adaptor-ligated fragment libraries. Using a liquid-handling robot in conjunction with Carboxy Terminated Magnetic Beads, we labeled each library sample using a unique 6 bp DNA barcode, which allowed multiplex sample processing and sequencing of 32 libraries in a single run using Applied Biosystems' SOLiD sequencer. We applied our semi-automated pipeline to targeted medical resequencing of nuclear candidate genes in individuals affected by mitochondrial disorders. This novel method is capable of preparing as much as 32 DNA libraries in 2.01 days (8-hour workday) for emulsion PCR/high throughput DNA sequencing, increasing sample preparation production by 8-fold

Regulation of Toll-like Receptor Signaling by the SF3a mRNA Splicing Complex

<div><p>The innate immune response plays a key role in fighting infection by activating inflammation and stimulating the adaptive immune response. However, chronic activation of innate immunity can contribute to the pathogenesis of many diseases with an inflammatory component. Thus, various negatively acting factors turn off innate immunity subsequent to its activation to ensure that inflammation is self-limiting and to prevent inflammatory disease. These negatively acting pathways include the production of inhibitory acting alternate proteins encoded by alternative mRNA splice forms of genes in Toll-like receptor (TLR) signaling pathways. We previously found that the SF3a mRNA splicing complex was required for a robust innate immune response; SF3a acts to promote inflammation in part by inhibiting the production of a negatively acting splice form of the TLR signaling adaptor MyD88. Here we inhibit SF3a1 using RNAi and subsequently perform an RNAseq study to identify the full complement of genes and splicing events regulated by SF3a in murine macrophages. Surprisingly, in macrophages, SF3a has significant preference for mRNA splicing events within innate immune signaling pathways compared with other biological pathways, thereby affecting the splicing of specific genes in the TLR signaling pathway to modulate the innate immune response.</p></div

Plan to investigate the effect of SF3a1 inhibition.

<p>The schematic depicts our experimental approach for investigating the effect of SF3a1 inhibition on mRNA splicing and innate immunity. Mouse RAW264.7 cells were treated with either SF3a1 siRNA or control siRNA and subsequently exposed (or not) to LPS. These mRNA samples were then subjected to poly-A mRNA sequencing (A). The sequence data were then analyzed using MISO (B) to determine the global effects of SF3a1 on alternative pre-mRNA splicing and to investigate intron sequences that mediated these alternative splicing effects (C). The sequence data also were analyzed using DESeq, DEXSeq, and Cuffdiff to identify genes and isoforms whose expression was regulated by SF3a1 (B). This gene- and isoform-level analysis was in turn used for pathway analysis (D) and to identify specific TLR signaling pathway genes that mediate the effects of SF3a on innate immunity (E).</p

SF3a1 inhibition leads to intron retention in several TLR signaling pathway genes.

<p>(A,B) These panels depict sequence reads (average of 3 replicates, each; generated by DEXSeq) across IRAK1 or IKKβ (5’ end of gene on right, control siRNA in red, SF3a1 siRNA in blue). The retained introns (intron 1 in IRAK1 or intron 15 in IKKβ) are shaded in purple. (C-I) These panels display the results of qPCR assays on RAW264.7 cells used to monitor production of the indicated mRNA isoforms (expression normalized so that 1 is the expression in the presence of control siRNA). CT indicates control siRNA in this an all other figures. LPS exposures were performed for six hours in the presence of 20 ng/ml LPS. Asterisks indicate results that were statistically different than control in this and all other figures.</p

Multiple genes in the TLR signaling pathway mediate the effects of SF3a1 on innate immunity.

<p>RAW264.7 macrophages were treated with the indicated siRNAs or control non-targeting siRNA and subsequently exposed to 20 ng/ml LPS for 6 hours. IL-6 production was monitored by ELISA. In panels B and C, cells were treated with multiple siRNAs simultaneously, as indicated; in panel A, only a single siRNA was used in each case.</p

siRNAs targeting IKKβ intron 15 lead to increased production of LPS-induced IL-6.

<p>RAW264.7 macrophages were treated with either of 2 siRNAs targeting IKKβ intron 15 or control non-targeting siRNA, were subsequently exposed to 20 ng/ml for 6 hours, and then expression of both IKKβ isoforms was monitored by qPCR (A,B) and IL-6 production was monitored by ELISA (C).</p

LPS challenge and SF3a1 inhibition affect pre-mRNA splicing of a common set of genes.

<p>The Venn diagram (generated using Vennerable package for R 3.0.1) indicates the number of genes whose splicing is altered in the DEXSeq analysis following LPS challenge alone, SF3a1 inhibition in the absence of LPS, or SF3a1 inhibition in the presence of LPS.</p

Identification of intron and exon features that correlate with alternative splicing events regulated by SF3a1.

<p>(A-D) Sequence logo plots of the polypyrimidine tracts and 3’ splice site of introns that are retained when SF3a1 is inhibited (SF3a-dependent) or introns that are spliced out correctly when SF3a1 is inhibited (SF3a-independent, or at least less dependent) in the RAW264.7 cell line. (E) The length of exons that are skipped when SF3a1 is inhibited compared to those exons that are not skipped. The boxplots were generated using default conventions in R 3.0.1. The boxes show the interquartile range, the whiskers the most extreme data point that is no more than 1.5 times the interquartile range from the box. Exon lengths are depicted on a log scale. “Skipped” refers to exons with increased rate of skipping when SF3a1 is inhibited, whereas “not skipped” refers to exons that have the same rate of skipping, regardless of SF3a1 levels.</p

SF3a1 regulates multiple modes of alternative pre-mRNA splicing.

<p>The figure depicts the percentage of possible alternative splicing events (intron retention, exon skipping, alternate 3’ and 5’ splice site choice, and mutually exclusive exon usage) affected by either LPS treatment or SF3a1 inhibition (in the absence or presence of LPS) in the RAW264.7 cell line. Complete data depicted in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004932#pgen.1004932.s021" target="_blank">S19 Table</a>.</p