Skip to main content
Fig. 2 | Genome Biology

Fig. 2

From: PHLI-seq: constructing and visualizing cancer genomic maps in 3D by phenotype-based high-throughput laser-aided isolation and sequencing

Fig. 2

Collection efficiency and the probability of obtaining high-quality sequencing data after cell isolation by PHLI-seq. a HL60 cells were prepared on ITO-coated glass slide, and single cells were isolated into separate tubes (n = 53). MDA reaction was carried out and monitored using real-time PCR machine. After the reaction, amplification start times (AST) were determined to quantify amplification qualities for each tube. We found that negative controls or failed single-cell isolations have ASTs over 50, while successful experiments have AST under 40. Then, we performed similar experiments with an H&E-stained breast cancer tissue section. From the 10-um tissue section, five cells or single-cell were isolated and amplified with 16 replicates, respectively. A portion of the amplified products is sequenced by low-depth whole-genome sequencing. b To quantify amplification uniformity, we used Pearson correlations between CNA profiles of the samples and the tumor bulk DNA (left). A sample with high correlation value shows similar CNA profile with that of the tumor bulk DNA, while samples with low correlations present profiles with large deletions and variations. c–e To analyze genomic coverage, area under Lorenz curve (AUC, the higher AUC is, the higher genomic coverage is) was calculated for each sample. We found that the AUC value was inversely related to the AST. Similarly, Pearson correlation value tends to be high, when AST is low. Finally, we set a threshold value (blue dashed line) to define high-quality amplification products, which guarantees high Pearson correlation with tumor bulk DNA (i.e., amplification uniformity), and high AUC value (i.e., high genome coverage). f, g To test whether irradiating IR laser pulse and vaporizing discharging layer generate DNA fragmentation, HL60 single cells were isolated and sequenced by PHLI-seq method. Single cells were isolated by pipetting or by PHLI-seq method with 1, 10, and 50 IR laser pulse in total. Isolated cells were amplified by MDA, and six samples from each group were sequenced. For comparison, 20 cells from formalin-fixed HL60 sample and gDNA extract from a population of HL60 cells were sequenced. MDA reaction produces uniform amplification profile and high genomic coverage when input DNA is long (not fragmented) and undamaged. For example, the result from formalin-fixed sample showed low uniformity throughout the genome because the formalin fixation method is known to generate DNA fragmentation. On the other hand, the other results showed more uniform profile than that of the formalin-fixed cells. Also, the cells which were isolated by PHLI-seq technique showed similar uniformity with those isolated by pipetting, suggesting that there is no sign of laser-induced damage

Back to article page