Qualitative and quantitative assessment of Illumina’s forensic STR and SNP kits on MiSeq FGx™

<div><p>Massively parallel sequencing (MPS) is a powerful tool transforming DNA analysis in multiple fields ranging from medicine, to environmental science, to evolutionary biology. In forensic applications, MPS offers the ability to significantly increase the discriminatory power of human identification as well as aid in mixture deconvolution. However, before the benefits of any new technology can be employed, a thorough evaluation of its quality, consistency, sensitivity, and specificity must be rigorously evaluated in order to gain a detailed understanding of the technique including sources of error, error rates, and other restrictions/limitations. This extensive study assessed the performance of Illumina’s MiSeq FGx MPS system and ForenSeq™ kit in nine experimental runs including 314 reaction samples. In-depth data analysis evaluated the consequences of different assay conditions on test results. Variables included: sample numbers per run, targets per run, DNA input per sample, and replications. Results are presented as heat maps revealing patterns for each locus. Data analysis focused on read numbers (allele coverage), drop-outs, drop-ins, and sequence analysis. The study revealed that loci with high read numbers performed better and resulted in fewer drop-outs and well balanced heterozygous alleles. Several loci were prone to drop-outs which led to falsely typed homozygotes and therefore to genotype errors. Sequence analysis of allele drop-in typically revealed a single nucleotide change (deletion, insertion, or substitution). Analyses of sequences, no template controls, and spurious alleles suggest no contamination during library preparation, pooling, and sequencing, but indicate that sequencing or PCR errors may have occurred due to DNA polymerase infidelities. Finally, we found utilizing Illumina’s FGx System at recommended conditions does not guarantee 100% outcomes for all samples tested, including the positive control, and required manual editing due to low read numbers and/or allele drop-in. These findings are important for progressing towards implementation of MPS in forensic DNA testing.</p></div

DUX4 Differentially Regulates Transcriptomes of Human Rhabdomyosarcoma and Mouse C2C12 Cells

<div><p>Facioscapulohumeral muscular dystrophy (FSHD) is linked to the deletion of the D4Z4 arrays at chromosome 4q35. Recent studies suggested that aberrant expression of double homeobox 4 (<i>DUX4</i>) from the last D4Z4 repeat causes FSHD. The aim of this study is to determine transcriptomic responses to ectopically expressed DUX4 in human and mouse cells of muscle lineage. We expression profiled human rhabdomyosarcoma (RD) cells and mouse C2C12 cells transfected with expression vectors of <i>DUX4</i> using the Affymetrix Human Genome U133 Plus 2.0 Arrays and Mouse Genome 430 2.0 Arrays, respectively. A total of 2267 and 150 transcripts were identified to be differentially expressed in the RD and C2C12 cells, respectively. Amongst the transcripts differentially expressed in the RD cells, <i>MYOD</i> and <i>MYOG</i> (2 fold, p<0.05), and six <i>MYOD</i> downstream targets were up-regulated in RD but not C2C12 cells. Furthermore, 13 transcripts involved in germline function were dramatically induced only in the RD cells expressing DUX4. The top 3 IPA canonical pathways affected by DUX4 were different between the RD (inflammation, BMP signaling and NRF-2 mediated oxidative stress) and the C2C12 cells (p53 signaling, cell cycle regulation and cellular energy metabolism). Amongst the 40 transcripts shared by the RD and C2C12 cells, <i>UTS2</i> was significantly induced by 76 fold and 224 fold in the RD and C2C12 cells, respectively. The differential expression of <i>MYOD, MYOG</i> and <i>UTS2</i> were validated using real-time quantitative RT-PCR. We further validated the differentially expressed genes in immortalized FSHD myoblasts and showed up-regulation of <i>MYOD</i>, <i>MYOG</i>, <i>ZSCAN4</i> and <i>UTS2</i>. The results suggest that DUX4 regulates overlapped and distinct groups of genes and pathways in human and mouse cells as evident by the selective up-regulation of genes involved in myogenesis and gametogenesis in human RD and immortalized cells as well as the different molecular pathways identified in the cells.</p></div

Heat map of iSNP genotype analysis for all samples and loci.

<p>Loci in columns follow the same order as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187932#pone.0187932.g001" target="_blank">Fig 1</a> for iSNPs (94). Reaction samples are in rows. Experiments and positive controls (2800M) are boxed. Color code: <b>green</b>–correct genotype; <b>light green</b>–editable genotype (see text); <b>yellow</b>–additional C-allele; <b>red</b>–genotype error; <b>gray</b>–ADO; <b>black</b>–LDO.</p

Overview of experiments.

<p>Nine experimental runs were performed with a total of 336 samples: 314 reaction samples, 9 NTCs, and 13 positive controls (2800M). Expt. I is the benchmark.</p

Heat map of read numbers for all samples and loci.

<p>The read numbers are shown in color code (low, medium, and high in blue, yellow, and red, respectively) for each sample and locus. Loci shown in columns: <b>aSTRs (28):</b> AMEL, TPOX, D3S1358, FGA, D5S818, CSF1PO, D7S820, D8S1179, THO1, vWA, D13S317, D16S539, D18S51,D21S11, D1S1656, D2S441, D2S1338, D4S2408, D6S1043, D9S1122, D10S1248, D12S391, Penta E, D17S1301, D19S433, D20S482, Penta D, D22S1045; <b>Y-STRs (24):</b> DYF387S1, DYS19, DYS385a-b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS437, DYS438, DYS439, DYS448, DYS460, DYS481, DYS505, DYS522, DYS533, DYS549, DYS570, DYS576, DYS612, DYS635, DYS643, Y-GATA-H4; <b>X-STRs (7):</b> DXS10074, DXS10103, DXS10135, DXS7132, DXS7423, DXS8378, HPRTB; <b>iSNPs (94):</b> rs1490413, rs560681, rs1294331, rs10495407, rs891700, rs1413212, rs876724, rs1109037, rs993934, rs12997453, rs907100, rs1357617, rs4364205, rs2399332, rs1355366, rs6444724, rs2046361, rs279844, rs6811238, rs1979255, rs717302, rs159606, rs13182883, rs251934, rs338882, rs13218440, rs1336071, rs214955, rs727811, rs6955448, rs917118, rs321198, rs737681, rs763869, rs10092491, rs2056277, rs4606077, rs1015250, rs7041158, rs1463729, rs1360288, rs10776839, rs826472, rs735155, rs3780962, rs740598, rs964681, rs1498553, rs901398, rs10488710, rs2076848, rs2107612, rs2269355, rs2920816, rs2111980, rs10773760, rs1335873, rs1886510, rs1058083, rs354439, rs1454361, rs722290, rs873196, rs4530059, rs1821380, rs8037429, rs1528460, rs729172, rs2342747, rs430046, rs1382387, rs9905977, rs740910, rs938283, rs8078417, rs1493232, rs9951171, rs1736442, rs1024116, rs719366, rs576261, rs1031825, rs445251, rs1005533, rs1523537, rs722098, rs2830795, rs2831700, rs914165, rs221956, rs733164, rs987640, rs2040411, rs1028528. Reaction samples are in rows. Experiments are boxed. Note, female samples did not show any read numbers at the Y-STRs and were kept in white. Locus drop out (LDO) are marked in black. <b>Positive controls (2800M)</b> of each experiment are shown at the bottom (kept in the same experimental order as in the figure; for Expt. IV a total of 5 positive controls were included).</p

Heat map of STR sequence analysis for all samples and loci.

<p>Loci in columns follow the same order as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187932#pone.0187932.g001" target="_blank">Fig 1</a> for aSTRs (28), Y-STRs (24), and X-STRs (7). Reaction samples are in rows. Experiments and positive controls (2800M) are boxed. Color code: <b>red</b>–genotype error not flagged by UAS; <b>orange</b>–genotype error flagged by UAS; <b>pink</b>–typed stutter plus typed sequence error (<b>SE</b>); <b>purple</b>–typed SE; <b>yellow</b>–typed stutter; <b>black</b>–locus drop-out (<b>LDO</b>); <b>gray</b>–untyped stutter; <b>turquoise</b>–untyped SE plus untyped SE from stutter; <b>light blue</b>–untyped SE; and <b>green</b>–no artifacts. Note, female samples did not show sequences at Y-STRs and were kept in <b>white</b> (except ADIs, see text). The <b>white</b> spacing between a-, Y-, and X-STRs separates the STRs.</p

Heat map of ACRs for all samples and loci.

<p>Loci in columns follow the same order as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187932#pone.0187932.g001" target="_blank">Fig 1</a> for aSTRs (28), Y-STRs (2): DYF387S1 and DYS385a-b; X-STRs (7), and iSNPs (94). Reaction samples are in rows. Experiments and positive controls (2800M) are boxed. Color code: <b>green</b>–ACR ≥0.7; <b>yellow</b>–ACR between 0.5–0.7; <b>orange</b>–ACR between 0.3–0.5; <b>red</b>–ACR ≤0.3; <b>white</b>–female samples at Y-STRs, male samples at X-STRs, and homozygotes that did not show an ACR; <b>gray</b>–ADO; and <b>black</b>–LDO.</p

Effects of experimental conditions on read numbers.

<p>Effects of experimental conditions on read numbers.</p

Sensitivity, dropouts, and ACR.

<p>Sensitivity, dropouts, and ACR.</p