Improving the Power of Structural Variation Detection by Augmenting the Reference

<div><p>The uses of the Genome Reference Consortium’s human reference sequence can be roughly categorized into three related but distinct categories: as a representative species genome, as a coordinate system for identifying variants, and as an alignment reference for variation detection algorithms. However, the use of this reference sequence as simultaneously a representative species genome and as an alignment reference leads to unnecessary artifacts for structural variation detection algorithms and limits their accuracy. We show how decoupling these two references and developing a separate alignment reference can significantly improve the accuracy of structural variation detection, lead to improved genotyping of disease related genes, and decrease the cost of studying polymorphism in a population.</p></div

An illustrative example.

<p>In the top scenario, a VNA (shown in red) is present in the donor. In ref+, only concordant alignments (correct orientation and mapped distance) are present. As a result, the SV caller does not make a call in ref+, which is converted by TRANSLATE_CALLS to an insertion call in the GRC reference (hg18). In the GRC reference, however, the read pairs that originate from across the VNA junction map discordantly, with one read left unmapped or falsely mapping to a homologous region. These signals in the GRC reference are difficult to decipher for any SV algorithm. In the bottom scenario, where the VNA is absent in the donor, the pairs that span the VNA injection point in the donor align concordantly to the GRC reference. In ref+, they align discordantly with an enlarged mapped distance but bear the hallmark signature of a deletion. This is among the easiest signals that an SV caller can detect and most algorithms show good results with respect to this SV type</p

Analysis of ref+ pipeline accuracy.

<p>Each vertical line represents one individual, with the plus (+) point representing the ref+ pipeline and the square point representing the GRC pipeline.</p

Method workflow.

<p><b>a)</b> In a traditional SV calling pipeline the reads are first aligned against the GRC reference and the alignments are passed to an SV caller, which annotates regions of the GRC reference as being inserted/deleted. <b>b)</b> Our approach is composed of two additional components. BUILD_REF takes a set of sequences to be inserted and modifies the GRC reference genome (e.g. hg18) by inserting the sequences into their prescribed locations, obtaining a new genome (ref+). We next align the reads to ref+ and run a SV caller. The TRANSLATE_CALLS component then modifies the resulting calls so that they become calls relative to the GRC reference, not ref+.</p

<i>Vav</i>-CAR mice have a reduced cellular number in their spleens, thymus and lymph nodes.

<p>The <b>(A, C)</b> spleens, <b>(B, D)</b> thymus and (<b>E</b>) inguinal lymph nodes of <i>vav</i>-CAR and wild type (WT) mice were harvested and weighed. Organs were dissociated into a single cell suspension and counted. Data represents the mean ± SEM of (Spleen WT = 13, F9 = 19, F38 = 18)(Thymus WT = 4, F9 and F38 = 8)(Lymph node WT = 4, F9 and F38 = 6)) mice. *p = 0.02, **p = 0.001, ***p ≤ 0.0004, ****p < 0.0001.</p

Transgene integration site for F9.

<p>Adapted from the screenshots from the IGV genome viewer show the integration sites of the transgene in chromosome 10. The colored reads indicate that the other half of the fragment maps discordantly. The teal colored reads are those that have mates on the transgene, and the purple reads (asterisked) on chr17 (the homologous segments to the transgene material, which are ambiguous for mapping purposes). Multi-colored segments at the ends of reads highlight soft-clipped portions of reads. There is a single cluster of soft-clipped reads for the F9 insertion site.</p

Multiple immune subsets express a CAR in the spleen, thymus and lymph node of <i>vav</i>-CAR mice.

<p>Splenocytes and lymphocytes derived from the thymus and lymph nodes of <i>vav</i>-CAR and WT mice were stained for surface markers (markers from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140543#pone.0140543.g004" target="_blank">Fig 4</a>) and chimeric antigen receptor expression. (<b>A, C, E, G, H</b>) Specific immune subsets were gated on to determine the percentage of CAR positive cells and (<b>B, D, F</b>) the mean fluorescence intensity (MFI) of CAR expression. Data represents the mean ± SEM of (WT = 4, F9 and F38 = 4–8) mice. **p<0.007, ****p<0.0001. SP: Single positive, DP: Double positive, DN: Double negative, Other: Negative for CD4, CD8, CD3 and TCRβ.</p

Schematic of <i>vav</i>-CAR plasmid generation (A) The HS21/45 plasmid containing the <i>vav</i> promoter flanking the transgene human CD4 and (B) the pCR4 TOPO cloning vector containing the chimeric antigen receptor transgene were digested using the restriction enzymes <i>Not1</i> and <i>Sfi1</i>.

<p>The HS21/45 plasmid and the CAR insert were then ligated and subjected to sequencing to verify the correct orientation. (<b>C</b>) The <i>vav</i>-CAR plasmid was then prepared using an endotoxin free kit, digested with <i>HindIII</i> and (<b>D</b>) the transgene microinjected into C57BL/6 fertilized oocytes. Not to scale.</p

The number of <i>vav-</i>CAR transgenes affects both proportion and expression level of CAR⁺ cells.

<p>Spleens from homozygous and heterozygous <i>vav</i>-CAR mice were harvested and analyzed for the level of receptor expression on resting T lymphocytes. Splenocytes were incubated with Fc receptor block and stained with the following antibodies; TCRβ, CD3, CD4, CD8 and Myc-tag Alexa488. The mean fluorescence intensity (MFI) of CAR expression on F9 mice <b>(A)</b> and F38 mice <b>(B)</b> is depicted. <b>(C)</b> Depicts the percentage of CD4⁺ and CD8⁺ subsets in spleens and the percentage of those subsets expressing CAR in homozygous (Hom) and heterozygous (Het) mice. Data represents the mean ± SEM of (WT = 2, Homozygous = 2, Heterozygotes = 7) individual mice. ****p<0.0001, NS = not significant.</p

CAR expression is varied between different immune subsets and founders.

<p>Splenocytes from <i>vav-</i>CAR (black line) and WT (grey line) mice were stained for surface markers (markers from <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140543#pone.0140543.g004" target="_blank">Fig 4</a></b>) and CAR expression. Individual immune subsets were gated on and the level of receptor assessed comparative to WT splenocytes. Representative FACS plot shown for each subset.</p