Long Span DNA Paired-End-Tag (DNA-PET) Sequencing Strategy for the Interrogation of Genomic Structural Mutations and Fusion-Point-Guided Reconstruction of Amplicons

10.1371/journal.pone.0046152PLoS ONE79

DNA-PET library construction, sequencing and mapping.

<p>(A) The genomic DNA was randomly sheared to different size range. (B) The very narrow region DNA fragments were obtained after size selection. (C) The purified DNA fragments were circularized, <i>EcoP15I</i> digested, sequencing adaptor ligated, and finally sequenced by SOLiD sequencer. (D) PET mapping span distribution of 1 kb (blue), 10 kb (red) and 20 kb (green) libraries. Based on the mapping pattern, PETs can be distinguished as concordant PETs and discordant PETs.</p

Statistics of PET sequencing.

1)<p>non-redundant.</p>2)<p>physical coverage.</p>3)<p>concordant PET.</p>4)<p>proportion of cPET to total NR PET.</p>5)<p>discordant PET.</p>6)<p>proportion of dPET to total NR PET.</p>7)<p>number of dPET involved in the dPET clusters with cluster count ≥3.</p

SV identification based on the mapping pattern of dPET clusters.

<p>The dark red and pink arrows represent the 5′ and 3′ anchor regions of the dPET cluster, respectively. Black, white and blue horizontal lines represent chromosome segments. The red track represents the coverage of cPETs. The dotted lines indicate the connections between the two dPET clusters. The sub-types of insertions are as follows: (1) Intra-chromosomal direct forward insertion. (2) Intra-chromosomal direct backward insertion. (3) Intra-chromosomal inverted forward insertion. (4) Intra-chromosomal inverted backward insertion. (5) Deletion plus intra-chromosomal direct forward insertion. (6) Deletion plus intra-chromosomal inverted forward insertion. (7) Inter-chromosomal direct insertion. (8) Inter chromosome inverted insertion.</p

Reconstruction of the <i>BCR-ABL1</i> amplicon of K562.

<p>(A) Concordant tag distributions representing copy number are shown for amplified genomic regions (top, green track). Genomic segments between predicted breakpoints are indicated by colored arrows and dPET clusters with cluster sizes greater than 35 of predicted somatic rearrangements are represented by horizontal lines flanked by dark red and pink arrows indicating 5′ and 3′ anchor regions (middle). Small to large dPET clusters are arranged from top to bottom. Cluster sizes are indicated. High dPET cluster size of the CML causing <i>BCR-ABL1</i> translocation suggests that the rearrangement occurred early and that it has subsequently been amplified. Fusion points I–III correspond to panels C–D. (B) Fluorescence <i>in situ</i> hybridization (FISH) of <i>BCR-ABL1</i> rearrangement (fusion point I with cluster size 692). Yellow spots represent fusion signals and illustrate the amplification of <i>BCR-ABL1</i>. (C) FISH analysis of metaphase chromosomes of three high copy fusion points: I) probes used in B show fusion signals on two marker chromosomes and on chromosome 2q and normal localization on both rearranged chromosomes 9 and normal chromosome 22; the fusion on chromosome 2 has not been identified by DNA-PET most likely due to low sequence complexity at the break point or complex rearrangements, II) probes spanning the fusion point II (cluster size 259) show fusion signals on the same marker chromosomes and normal localization on both normal and rearranged chromosomes 9 and 13, III) probes spanning fusion point III (cluster size 218) show fusion signals on the same marker chromosomes and normal localization on both normal chromosome 22 and rearranged chromosomes 9. (D) Contigs (indicated by boxes) which were covered by PET mapping were concatenated by fusion-point-guided-concatenation method. The length of a contig is represented by the length of the box. Because of the size difference between chromosomes 1, 3, 9, 13, and 22, the length of chromosome 22 is represented by the length of contig/10,000 while the lengths of chromosomes 1, 3, 9, and 13 are represented by the length of contig/100,000. Any value less than 0.1 is rounded to 0.1; any value larger than 6 is rounded to 6. The thickness of borders of each contig represents the coverage (copy number). Red dashed edges represent dPET edges, while black bold edges represent cPET edges. The thickness of dPET edges represents the size of the corresponding dPET cluster. cPET edges have uniform thickness. Arrow heads pointing towards a contig indicate connections with the lower coordinates, arrow heads pointing away from a contig indicate connections with the higher coordinates.</p