Tumor samples and DNA extraction

Archived FFPE pathology blocks of Merkel cell carcinoma (MCC) samples (n = 2) were obtained as previously described [6]. MCC cells from these previously analyzed samples were newly micro-dissected by the Roche Automated Tissue Dissection System (Roche) from 2–3 5-μm hematoxylin and eosin (H&E) stained sections, followed by shearing with sonication with the Covaris LE220 system. DNA was extracted using a MagAttract® HMW DNA mini Kit (Qiagen).

FFPE breast tumor samples (n = 4) were obtained for this study from the LifePool cohort (www.lifepool.org). LifePool prospectively recruits Australian female participants through the population-based mammographic screening program. Participants consent to use of their diagnostic tissue blocks for research. Ten-micron sections were H&E stained and DNA was extracted from manually needle micro-dissected cells using the Qiagen DNeasy FFPE Kit (Qiagen) as previously described [11] from both FFPE breast tumor samples and two FFPE pre-cancerous breast lesions (papilloma). The quality of DNA was assessed by a multiplex PCR assay [12] modified to include additional primer sets that produce up to 700 bp fragments from non-overlapping target sites in the GAPDH gene.

This study was approved by the Human Research Ethics Committee at the Peter MacCallum Cancer Centre. This study was carried out in accordance with all relevant regulations and guidelines.

Whole genome amplification

Extracted DNA from FFPE MCC samples were amplified using GenomePlex® Complete Whole Genome Amplification (WGA) kit (Sigma-Aldrich), following the manufacturer’s instruction with several minor modifications. In brief, 50 ng of DNA was prepared in a total volume of 10 μL for the fragmentation, followed by library preparation and 14 cycles of amplification as described in the protocol. The final product was purified using QIAquick® PCR purification kit (Qiagen), followed by quantification to determine the final concentration; the yield was 2–4 μg. The average fragmentation size of WGA products was 200–300 bp. A standard human genomic DNA was used as a positive control provided with the Genome Plex WGA kit (Sigma) and a no template control was used as a negative control.

NEB next FFPE repair

The NEB Next FFPE Repair kit (NEB M6630, New England® Biolabs Inc) was used for repairing 150 ng of total DNA, according to the manufacturer’s protocol with a minor change of eluting DNA in 30 μL instead of 40 μL. A total of 10 μL of eluted DNA (total 50 ng of repaired DNA) was used for WGA using the Sigma WGA kit as described above. The remaining 100 ng of repaired DNA was used for the library prep directly using the KAPA Hyper Prep Kit.

KAPA Hyper library preparation

Library preparation was performed as described in the KAPA Hyper Prep Kit Illumina® platforms (KR0961-v1.14, KAPA Bio systems). Slight modifications of the manufacturer’s protocol were incorporated. Briefly, 100 ng of non-WGA or unamplified DNA (both NEB Next treated repaired DNA and untreated DNA) was sheared with sonication (Covaris S2 system) for 3 × 60 s, with the following parameters: duty cycles of 10, intensity of 5, and 200 cycles/burst.

Subsequently, libraries of the both the fragmented unamplified DNA (200–400 bp) and WGA products were created by end repair and A-tailing, adaptor ligation with a stock concentration of 15 μM adaptor, followed by library amplification of six PCR cycles and eluted in 30 μL after post-amplification clean up. The library distribution was analyzed by TapeStation 2200 (Agilent Technologies) and quantified by Qubit (Life Technologies).

NEBNext® Ultra ™ II DNA Library Prep

Library preparation was performed from MCC samples (n = 2) with 5 ng and 20 ng input, breast tumor samples (n = 4), and pre-cancerous breast lesions (papilloma) (n = 2) with 5 ng DNA input as described in the NEBNext® Ultra ™ II DNA Library Prep Kit (NEB E7645S/L, New England BioLabs ® Inc.) with several minor modifications. In brief, DNA fragmented using the Covaris S2 in 50 μL was used for NEBNext End Prep, followed by an immediate adaptor ligation step with a 1.5 μM diluted adaptor. Clean-up of adaptor-ligated DNA without size selection was carried out followed by PCR amplification with eight cycles and ten cycles for 20 ng and 5 ng input, respectively. After adding resuspended AMPure XP Beads to the PCR products, the mixture was incubated at room temperature for at least 20 min instead of 5 min. Subsequently, after adding 33 μL elution buffer (0.1 × TE) into the beads after washing with ethanol, it was incubated for 10 min instead of 2 min. A total of 2 μL of the final 30 μL library was analyzed with the TapeStation for the size distribution.

Low coverage whole genome sequencing

The libraries prepared by both KAPA Hyper and NEBNext kits were used for LC WGS. An Illumina Nextseq platform (NextSeq 500) (paired-end 75 bp, on a mid-output flow cell) was used to run the pooled, normalized indexed libraries according to the standard protocol. The final concentration was 2 nM pooled and diluted to 1.8 pM as the standard Illumina protocol. Sequencing of those samples led to genome coverage of 1.6–1.8 × per sample.

Molecular inversion probe SNP arrays

The Affymetrix Molecular Inversion Probe (MIP) 330 K OncoScan array was used to analyze four breast cancer samples (version 3) and two papilloma samples (version 2) and was performed according to the manufacturer’s instructions by the Ramaciotti Centre for Genomics (version 3, NSW, Australia) or Affymetrix Inc (version 2, Santa Clara, CA, USA). DNA input was 40–100 ng for this assay.

Data analysis

Reads were aligned with bwa mem (v0.7.12-r1039) [13] to hg19 (GRCh37) after removal of sequencing primers by cutadapt (v1.7.1) [14]. ControlFREEC (version 6.7) [15] was used to estimate copy number from the low-coverage WGS data in 50 kb windows across hg19, with default parameters, no matched normal sample and baseline ploidy set to 2. Down-sampling of bam files was performed with samtools [16].

MIP data were pre-processed by the Ramaciotti Centre for Genomics or Affymterix Inc., with tumor samples batch normalized against Affymetrix controls [11].

All sample data were imported into Nexus (BioDiscovery Inc., Hawthorne, CA, USA) and segmented using SNP-FASST, a circular binary segmentation algorithm. Copy number gains were called if the log₂ ratio of the segment was >0.15 and losses called if < –0.15. To reduce spurious calls, the genome was masked using a list of published problematic regions, including highly repetitive centromeric regions, where DNA copy number cannot be accurately measured [8].

Total CN profile overlap analysis was performed using Partek Genome Suite (Partek Inc., St. Louis, MO, USA). CNA segments for each matched pair were imported and the “finding regions in multiple samples” tool run, matching for event type (amplification/deletion). This tool reports each CNA region shared at base-pair resolution as well as each CNA region unique to a sample. Shared CN neutral regions were calculated by subtracting the length of all shared CNAs as well as sample only CNA events from the total base pairs covered.

Median Absolute Pair-wise Difference (MAPD) score was calculated as follows: if xi: is the log₂ ratio for marker i: then MAPD = median(|x _i+1  − x _i |,i ordered by genomic position). This metric provides a measure of the noise of the sample that is less dependent on true biological copy number variation than, for example, standard deviation.

FREEC normalized read counts in 50 kb bins were extracted from regions called as a gain or loss by FREEC in at least one of the 5 ng, 20 ng, and 100 ng DNA inputs or the WGA libraries for MCT-4 and MCT-6 LC WGS data. Gains or losses in regions lacking MIP array probes and regions in the blacklist of Scheinin et al. [8] were filtered out. The Pearson correlation of bin counts in these CNA regions was calculated and used to cluster by Euclidean distance using the hclust() function of R 3.2.1. Correlation between samples was visualized using the pheatmap package.

Comparing copy number alteration calls using low-input DNA

We investigated a recently developed library preparation method (NEBNext Ultra II) to reduce the required input of DNA (Fig. 1). DNA was obtained from two archival FFPE Merkel cell carcinoma (MCC) samples [6]. LC WGS was performed on 100 ng, 20 ng, and 5 ng input DNA. Compared with the standard 100 ng input, comparable CN profiles were observed using 5 ng or 20 ng input DNA with 95 % of CN calls (gain, loss, or no change) being concordant on average (Figs. 2 and 3). In addition, the quality control metric MAPD was comparable between the different DNA inputs (Fig. 3).

Comparison of CN profiles from low-input LC WGS and MIP arrays

As the Affymetrix OncoScan MIP arrays are considered by many to be a high-quality method for CN analysis of FFPE samples [17], we compared the performance of low-input LC WGS against these SNP arrays using matched DNA from four FFPE breast cancer samples (LPS1-LPS4). The CN profiles derived from LC WGS were comparable to and, in some cases, improved upon MIP arrays (Fig. 4). Overall, LC WGS with 5 ng of input DNA resulted in CN profiles with >80 % (80–93 %) concordance with those produced using 80–100 ng input DNA on MIP arrays (Table 2). LC WGS typically covers 60–80 % of the sites in hg19 (Table 1), providing broader sampling of the genome, apart from the genomic regions known to be problematic for CN estimation [8], than MIP arrays, which interrogate ~330,000 selected sites that may not be distributed evenly across the genome.

Sample	Shared sites (%)	WGS only sites (%)	CNA WGS only (Mbp)	MIP only sites (%)	CNA MIP only (Mbp)	Shared CNV (%)
LPS1	80.0	10.1	263	9.9	260	68
LPS2	83.4	7.7	202	8.8	231	74
LPS3	83.0	9.6	198	7.4	136	72
LPS4	93.1	6.6	173	0.34	8.9	49

It is noteworthy that from the overlap analysis (Fig. 3), on average 15 % of the total CN profiles from LC WGS and MIP differed; these differences fell into two categories. First, in some cases, LC WGS provided higher sensitivity to detect small CN changes by providing more even coverage across the genome than SNP arrays, whereas, in other regions where SNP density was high, the MIP arrays were able to detect CNA with length <50 kb, below the detection limit imposed by the chosen window size for LC WGS analysis. Second, many of the large-scale differences were caused by segmentation and thresholding differences, rather than true CN changes (Additional file 1: Figure S1 and S2). For example, in LP S1, MIP arrays called chromosome 4 as a loss whereas no CNA was called from the LC WGS data (Additional file 1: Figure S2). However, that particular loss could be explained by some segments sitting just below the threshold in the MIP data whereas in LC WGS bins they did not, due to normalization subtly shifting read counts upward across the genome. For three samples, we had orthogonal CN data from a targeted sequencing assay. Good concordance was observed for CN variable regions between LC WGS and this assay (LPS1 84 %, LPS2 94 %, and LPS4 61 % of CNA bp concordant). The concordance between MIP arrays and the targeted assay was similar for CNA regions (87 %, 95 %, and 61 %, respectively).

We also compared the performance of low-input LC WGS against MIP arrays using matched DNA from two FFPE pre-cancerous breast lesions in order to investigate whether LC WGS could offer an improvement upon very poorly performed MIP assays. Both low-quality DNA LC WGS samples demonstrated improved resolution of CNAs as compared with MIP arrays (Fig. 5). P1 showed markedly improved segmentation continuity, with 1306 segments resolving into 68 segments. Sample P2 showed a particularly big reduction in bin-to-bin variability (Additional file 1: Figure S3) and the proportion of data points greater than twice the mean CNA value reduced from 17 % to just 1 %.

The FFPE repair treatment made little discernible difference to the appearance of the CN profiles (data not shown) or MAPD scores (Fig. 3), although the sequencing metrics were marginally improved compared with untreated samples (Table 1).

Comparison of CN profiles between unamplified and WGA samples with or without NEB FFPE repair treatment

We additionally evaluated WGA as an alternative method of reducing the amount of native input DNA into LC WGS without compromising CNA detection. In parallel, we assessed whether a DNA repair procedure (NEB Next) could improve LC WGS CNA detection performance. The experimental strategy is summarized in Fig. 1. Fifty nanograms of DNA derived from two archival FFPE MCC samples [6] were subjected to WGA and this yielded 2–4 μg of product, indicating that WGA was successful. LC WGS was performed on the same amount of input DNA (100 ng) from unamplified and WGA samples. Compared with the unamplified samples, the WGA samples had fewer reads mapped, approximately six times as many duplicate reads, and <15 % of the genome covered by at least one read (Table 1). The poorer sequencing metrics were reflected in the CN profiles with the unamplified samples showing less variability in read counts per genomic segment and more clearly discernible CNAs (Fig. 6). Further investigation revealed the poor sequencing results from the WGA samples are mostly likely related to base calling and read mapping being compromised by the presence of adaptors from WGA primers in the reads (Additional file 1: Figure S4).

Overall, 77–87 % of the total CN profiles from matched unamplified or WGA samples were concordant (Fig. 3). Unsupervised clustering of MCT-4 and MCT-6 CNA showed high intra-sample concordance with different input amounts and methods, although the WGA data had longer branch lengths (Additional file 1: Figure S5). Variance in read distribution as calculated by MAPD was much higher in WGA samples (Fig. 3), consistent with the higher level of noise observed in CN profiles of WGA samples as compared with unamplified. Down-sampling reads from the untreated unamplified samples to coverage equivalent to the WGA samples (0.2×) revealed the reduction in consistent CNA calls from the latter could not be attributed to differences in read depth alone (Fig. 3).