Journal: Heredity

Article Title: A new hybrid approach for MHC genotyping: high-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra)

doi: 10.1038/s41437-018-0070-5

Figure Lengend Snippet: Laboratory procedure used to obtain MHC II DRB short and long amplicons, and assembly pipelines. The short amplicon was sequenced by standard Sanger sequencing (step 1), while the long amplicon was sequenced with Illumina MiSeq and nanopore MinION after gel extraction (step 2). The gene structure is based on Bos taurus (Ensembl: ENSBTAG00000013919) and Ovis aries (GenBank AM884914) structures. Boxes: coding regions, lines: introns, horizontal arrows: PCR primers

Article Snippet: More in detail, we designed primers on DRB exons 1 and 3 and set-up a long-range PCR coupled with Illumina NGS sequencing to a very high coverage.

Techniques: Amplification, Sequencing, Gel Extraction, Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Validation of a novel Mho microarray for a comprehensive characterisation of the Mycoplasma hominis action in HeLa cell infection

doi: 10.1371/journal.pone.0181383

Figure Lengend Snippet: RT-PCR analysis of the postulated ABC transporter genes 730–760. The positions of the different amplicons are shown below the scheme of the MHO_710–770 gene region. The primers used ( Table 1 ) and the lengths of the amplicons (A-G) are indicated. PCR products (A–G) for genomic DNA (g), mRNA (m) and cDNA (c) were separated on a 0.6% agarose gel and stained with ethidium bromide. M, Gene Ruler 1 kb DNA ladder (Fermentas).

Article Snippet: Overlapping regions of the MHO genes 720–770 were amplified using the Long Range PCR Kit (Qiagen, Hilden, Germany) by standard PCR conditions (initial cycle of 3 min at 93°C; 35 cycles of 15 s at 93°C, 30 s at 50°C, 10 min at 68°C).

Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

Journal: Neural Development

Article Title: Interactions between innexins UNC-7 and UNC-9 mediate electrical synapse specificity in the Caenorhabditis elegans locomotory nervous system

doi: 10.1186/1749-8104-4-16

Figure Lengend Snippet: Expression patterns of other neuronal innexins . (A) Predicted gene structures of neuronal innexins. Arrows indicate primer binding sites used for PCR-amplified green fluorescent protein (GFP) constructs (see Materials and methods). (B) INX-1::GFP expression (green) in motor neurons; DAPI stained nuclei in red. (C) INX-1::GFP expression (green) in dorsal nerve cord (dnc) and body wall muscles (bwm); dorsal nerve cord is visualized with anti-UNC-33 antibody (red). (D) INX-19::GFP expression in AVA and AVB interneurons. (E) INX-4::GFP expression in sensory neurons and pharyngeal m1 muscle cell. Abbreviations: nr, nerve ring.

Article Snippet: unc-9 rescue and innexin::gfp constructions using long-range PCR A plasmid encompassing the unc-9 gene from exon 4 to the polyadenylation site at nucleotide 12,661 of cosmid R12H7 [ ] was constructed in pBluescript (Stratagene, Cedar Creek, TX, USA) extending from the Kpn I site at nucleotides 8,959–8,964 to the Xba I site at nucleotides 14,519–14,524.

Techniques: Expressing, Binding Assay, Polymerase Chain Reaction, Amplification, Construct, Staining, Antiviral Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: The human GRAF gene is fused to MLL in a unique t(5;11)(q31;q23) and both alleles are disrupted in three cases of myelodysplastic syndrome/acute myeloid leukemia with a deletion 5q

doi:

Figure Lengend Snippet: ( A ) Results of RT-PCR analyses. Lane M contains a size marker VI (Boehringer Mannheim). Lanes 1, 4, 7, and 12 are negative controls in which the cDNA was replaced by sterile water. Normal fragments are obtained from the patient's unaffected GRAF allele (lane 2) and from the cell line Mono-Mac6 (lane 3). A MLL / GRAF fusion mRNA is detected in the sample with a t(5;11)(q31;q23) (lane 5) but not in the cell line lacking this translocation (lane 6). Normal fragments are obtained from the patient's unaffected MLL allele (lane 8) and the cell line Mono-Mac6 (lane 9). The additional fragments in lanes 5, 8, and 9 are generated by MLL splice variants. Further analysis reveals that the reciprocal GRAF / MLL fragment is neither present in the patient's sample (lane 10) nor in the cell line (lane 11). Control amplifications with primers specific for the ABL gene are shown in lanes 15 (patient sample) and 16 (cell line). ( B ) Long-range PCR results of genomic DNA. Lanes M contain the size markers III and VI (Boehringer Mannheim). Lane 1 is a negative control. A MLL / GRAF fusion product is detected in the patient with the t(5;11)(q31;23) (lane 2) but not in the control cell line Mono-Mac6 (lane 3). Lanes 4, 5, 9, and 17 are negative controls. A normal 8-kb fragment that covers the breakpoint cluster region of the unaffected MLL alleles in the patient with the t(5;11)(q31;q23) (lane 6), in a healthy individual (lane 7), and in the Mono-Mac cell line (lane 8) is seen. No reciprocal GRAF / MLL gene fragment is detected in any of these samples (lanes 10–13), whereas in all of them an approximately 13-kb long intron of GRAF becomes evident (lanes 14–16). ( C ) Sequence and schematic representation of the inverted duplication of MLL within the genomic MLL / GRAF fusion. Numbering of nucleotides within the breakpoint region of MLL . The horizontal arrows indicate the positions of the primers used for amplification of the genomic MLL / GRAF fusion seen in lane 2 of B .

Article Snippet: After amplification of both the genomic unrearranged MLL and the MLL / GRAF fusion by long-range PCR we digested the PCR products by Dde I or Tru91 (Boehringer Mannheim).

Techniques: Reverse Transcription Polymerase Chain Reaction, Marker, Translocation Assay, Generated, Polymerase Chain Reaction, Negative Control, Sequencing, Amplification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: The human GRAF gene is fused to MLL in a unique t(5;11)(q31;q23) and both alleles are disrupted in three cases of myelodysplastic syndrome/acute myeloid leukemia with a deletion 5q

doi:

Figure Lengend Snippet: ( A ) Insertion of 52-bp (capital letters) derived from intron 13 into the final cDNA found in patient #7. The surrounding intronic sequences are shown in lowercase letters. This leads to a reading frame shift followed by a premature stop codon. The GAP domain of Graf is substantially shortened. The intronic regions that were sequenced in patient #7 and 12 healthy controls are indicated by arrows. The splice branch site consensus sequence is shown as follows: Y represents T or C, R either A or G. ( B ) Schematic representation of both cDNA fragments that were coamplified by universal primers I and IV for assessment of their relative amount. Primers II and III amplify the aberrantly spliced fragment only. ( C ) Nested PCR analysis using the first-round primers I and IV and the second-round primers II and III. M, molecular weight marker. Lane 1, negative control. Four of 15 healthy blood donors expressed the aberrantly spliced fragment in their mononuclear cells (lanes 2 and 4–6) because a faint PCR product was seen. Lane 12, positive control. ( D ) Single-round PCR analysis using primers I and IV. Lane 6, two differently sized PCR products are seen from the cDNA of patient #7 even after only one round of PCR. Positive plasmid controls containing the 52-bp insertion (lane 12) or not (lane 13). In each RT-PCR, 2 μg of total RNA was subjected to cDNA synthesis and processed in parallel.

Article Snippet: After amplification of both the genomic unrearranged MLL and the MLL / GRAF fusion by long-range PCR we digested the PCR products by Dde I or Tru91 (Boehringer Mannheim).

Techniques: Derivative Assay, Sequencing, Nested PCR, Molecular Weight, Marker, Negative Control, Polymerase Chain Reaction, Positive Control, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: Human Mutation

Article Title: Flexible and Scalable Full‐Length CYP2D6 Long Amplicon PacBio Sequencing

doi: 10.1002/humu.23166

Figure Lengend Snippet: Barcoding schemes. Direct versus two‐step sample barcoding. A : In the direct barcoding scheme the sample specific barcodes, indicated by the blue and red for patients 1 and 2, respectively, are attached to the gene‐specific sequences (black arrows) and are introduced in a single PCR reaction. B : For the two‐step procedure for each individual, the region of interest is first amplified with a pair of gene‐specific primers with M13 forward (green) and reverse (purple) sequence tails. A symmetrical sample barcode, indicated by blue and red for patients 1 and 2, respectively, is introduced in a second PCR using a set of M13 barcode primers. A 5' padding sequence (black) is present on the index primers for both the direct and two‐step barcoding schemes to give all fragment identical end sequences to avoid ligation biases during the SMRT bell library preparation.

Article Snippet: Long‐Range PCR, SMRT Library Prep, and PacBio Sequencing All work described in this paper is subject to the LUMC Good Research Practice & Integrity guidelines and Ethical requirements.

Techniques: Polymerase Chain Reaction, Amplification, Sequencing, Ligation

Journal: PLoS Genetics

Article Title: A Complex Structural Variation on Chromosome 27 Leads to the Ectopic Expression of HOXB8 and the Muffs and Beard Phenotype in Chickens

doi: 10.1371/journal.pgen.1006071

Figure Lengend Snippet: Analyzing the breakpoints of the copy number variations on GGA27 to clarify the rearrangement pattern. The duplicated regions identified by array-CGH are illustrated by green (CNV1), blue (CNV2) and pink (CNV3) boxes, respectively. (A) The boundaries of the CNVs were tested using 8 primers indicated by the arrows located in the known duplicated region of CNV1 and CNV3. All possible amplifications were considered and performed in both Mb and wild-type chickens. (B) The breakpoints of both CNV1_3’ (1,721,521 bp) and CNV3_5’ (4,470,331 bp) were identified after sequencing the specifically-amplified PCR product obtained in Mb chickens using primer F1 and R2. An overlap of two nucleotides was detected in the junction region. Outward facing primers (green and pink arrows) were designed to analyze the other boundary of CNV1 and CNV3. (C) CNV2 was found to be located next to CNV3 in chickens with the Mb phenotype using a genome-walking strategy. The breakpoints of both CNV3_3’ (4,503,417 bp) and CNV2_5’ (3,578,409 bp) were verified by sequencing. A two-nucleotide insertion was found in the junction region. (D) The breakpoints of both CNV2_3’ (3,592,890 bp) and CNV1_5’ (1,702,269 bp) were confirmed through unmapped read alignment of whole genome re-sequencing data. An eight-base insertion was detected in the junction.

Article Snippet: Re-sequencing of targeted genomic regions Re-sequencing of the three identified CNVs sequences was performed using long-range PCR and Ion Torrent Technology.

Techniques: Sequencing, Amplification, Polymerase Chain Reaction

Journal: BMC Microbiology

Article Title: Variation suggestive of horizontal gene transfer at a lipopolysaccharide (lps) biosynthetic locus in Xanthomonas oryzae pv. oryzae, the bacterial leaf blight pathogen of rice

doi: 10.1186/1471-2180-4-40

Figure Lengend Snippet: The lps locus is absent from the genomes of Xoo strains BXO8, Nepal624 and Xoor strain BXORI. (A) PCR analysis using primers that are specific to wxoA gene. M is the λ Hind III Marker lane. An expected band of 1 kb (indicated by arrow) is present in the Xoo strains, BXO1 (lane 1), BXO5 (lane 3), BXO6 (lane 4) and BXO20 (lane 6) but absent in BXO8 (lane 5), Nepal624 (lane 7) and BXORI (lane 2). (B) Southern hybridization analysis of Eco RI digested genomic DNA using α- 32 -P labeled wxoA specific probe (see Methods). A 4 kb band can be seen (indicated by arrow) in BXO1 (lane 1) but not in BXORI (lane 2), BXO8 (lane 3) and Nepal624 (lane 4). Similar results were obtained for wxoB , wxoC , wxoD , wzm and wzt genes. (C) The blot from (B) was deprobed and was hybridized with α- 32 P labeled probe specific to the metB gene. A specific band can be seen in all the lanes. Note the sizes of the bands indicating that metB is present in different Eco RI fragments in BXO1, BXORI and BXO8/Nepal624.

Article Snippet: Contig assembly was confirmed by restriction fragment analysis of a 12.5 kb PCR amplified product containing the lps locus that was obtained using long range PCR (Triple Master™, Eppendorf, Hamburg, Germany) with BXO1 genomic DNA as template.

Techniques: Polymerase Chain Reaction, Marker, Hybridization, Labeling

Journal: BMC Microbiology

Article Title: Variation suggestive of horizontal gene transfer at a lipopolysaccharide (lps) biosynthetic locus in Xanthomonas oryzae pv. oryzae, the bacterial leaf blight pathogen of rice

doi: 10.1186/1471-2180-4-40

Figure Lengend Snippet: Genetic organization of a locus encoding LPS biosynthetic genes in Xoo strain BXO1. a. Overall G+C content of the locus and the flanking regions. The G+C content of the genomic island was calculated without including the sequences of IS elements. The overall G+C content of the genome is ~65%. b. Organization and G+C content of individual genes and transposases of IS elements. IS 1114 encodes a truncated ORF. Arrows indicate transcriptional orientation. c. and d. Presence (+) and absence (-) of genes/PCR products in particular strains. § Indicates PCR products obtained using primer pairs directed against either metB and wxoA or etfA and wzm . # similar results were obtained with all Xoo strains tested excepting BXO8 and Nepal 624. * similar results were obtained with the Nepal 624 strain.

Article Snippet: Contig assembly was confirmed by restriction fragment analysis of a 12.5 kb PCR amplified product containing the lps locus that was obtained using long range PCR (Triple Master™, Eppendorf, Hamburg, Germany) with BXO1 genomic DNA as template.

Techniques: Polymerase Chain Reaction

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Noninvasive Immunohistochemical Diagnosis and Novel MUC1 Mutations Causing Autosomal Dominant Tubulointerstitial Kidney Disease

doi: 10.1681/ASN.2018020180

Figure Lengend Snippet: MUC1 VNTR sequencing identifies novel mutations causing ADTKD- MUC1 . (A) Sequence logo showing the most conserved regions of the VNTR repeats. Corresponding amino acid sequences of wild-type MUC1 (wt_AA) and MUC1 fs (mut_AA) are shown below. To find novel frameshift mutations that change the open reading frame, different conserved 10-mers of the wild-type repeat were used as sequence anchors (underlined DNA sequence as an example). For each anchor pair, all sequences delimited by these two anchors that are changing an open reading frame ( i.e. , adding or deleting nucleotides) were selected from the FASTQ file. (B) Sequences of the canonical 60 nucleotide long wild-type VNTR repeat (wt) and candidate frameshift mutations identified in this study. (C) Random mutations are generated in DNA molecules during PCR amplification step. To find true germline mutations, the percentage of reads with a given sequence (putative frameshift mutation) from all reads was calculated for each of the analyzed samples ( y -axis), and this needed to be higher than the average+2 SD of the nine wild-type control samples. Indicated are numbers of controls (wt), patients with individual MUC1 mutations (27dupC, 28dupA, 26_27insG, 1–16dup, 23delinsAT, 51dupC), and individuals with still unknown MUC1 mutation(s) who have urinary cell smears positive for MUC1fs and who tested negative for 27dupC by conventional genotyping assay (unknown). (D) 27dupC, confirmed by a mass spectrometry-based primer extension assay. The 27dupC extension product is observed at 5904 D (red asterisk). (E) 28dupA, confirmed by a mass spectrometry-based assay. The 28dupA extension product is observed at 6571 D (red asterisk). (F) 26_27insG, confirmed by a mass spectrometry-based assay. The 26_27insG extension product is observed at 5944.85 D (red arrow). (G) 1–16dup confirmed by restriction analysis. The mutation creates new restriction site for Eci I enzyme. The electrophoretogram shows amplified VNTR regions of the affected patient (P1), two healthy relatives (H1, H2), and one unrelated control (NC) after (EciI) and before restriction by Eci I (PCR). The patient’s (P1) mutated allele (5000 bp) was cut into two fragments of 3000 and 2000 bp. (H) 23delinsAT, confirmed by restriction analysis. The mutation creates new restriction site for Fok I enzyme. The electrophoretogram is showing amplified VNTR regions of two affected patients (P1, P2) and one unrelated control (NC) after (FokI) and before restriction by Fok I (PCR). The patients’ (P1, P2) mutated alleles (3000 bp) were cut into two fragments of 2000 and 1000 bp.

Article Snippet: These sequences were then read with the Illumina system (see the following references for an explanation of the Illumina system)., The VNTR region was directly amplified from genomic DNA using primers PS2F-T7 (5′-GGATCCTAATACGACTCACTATAGGAACAGACCACCATGGGAGAAAAGGAGACTTCGGCTACCCAG-3′) and PS3 (specified above) and long-range PCR (TaKaRa LA Taq DNA Polymerase with GC Buffers; Takara, Mountain View, CA).

Techniques: Sequencing, Generated, Polymerase Chain Reaction, Amplification, Mutagenesis, Genotyping Assay, Mass Spectrometry, Primer Extension Assay

Journal: Genes & Development

Article Title: Biological functions of the ISWI chromatin remodeling complex NURF

doi: 10.1101/gad.1032202

Figure Lengend Snippet: nurf301 is required for homeotic gene expression. ( A,B ) UBX protein in imaginal discs of the third thoracic segment (brown staining) is undetectable in nurf301 1 homozygotes. ( C,D ) Antibody staining shows that EN protein (revealed in green), which normally can be detected in the posterior compartment of all imaginal discs, is lost in nurf301 2 mutant larvae. ( E ) Semiquantitative RT–PCR analysis confirms that Ubx and en transcript abundance is reduced between 5- and 25-fold in total RNA isolated from nurf301 mutant animals. Lanes represent fivefold serial dilutions of mutant and wild-type total RNA (lane 1 , 200 ng; lane 2 , 40 ng; lane 3 , 8 ng; and lane 4 , 1.6 ng).

Article Snippet: EMS-induced nucleotide changes were determined by amplifying overlapping DNA fragments covering nurf301 using nurf301 -specific primers and an Expand Long Range PCR kit (Boehringer, Mannheim) and DNA isolated from homozygous mutant animals as template.

Techniques: Expressing, Staining, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Isolation

Journal: BMC Genomics

Article Title: Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulfite sequencing (SMRT-BS)

doi: 10.1186/s12864-015-1572-7

Figure Lengend Snippet: SMRT-BS validation. (A) Methylation quantitation by SMRT-BS of 42 CpG sites was compared to available data on the same sample using the HumanMethylation450 BeadChip (450K Array), resulting in an overall correlation of 0.906 ± 0.052. Correlation analyses were stratified by amplicon lengths, indicating a reduction in correlation with longer amplicons and possible PCR bias towards unmethylated DNA for the intermediate methylation region (see Results and discussion ). (B) Methylation quantitation by SMRT-BS of 174 CpG sites was compared to available data on the same sample using the SureSelect™ Human Methyl-Seq target enrichment next generation sequencing platform (MethylSeq), resulting in an overall correlation of 0.933 ± 0.031. Correlation analyses were stratified by amplicon lengths, indicating a reduction in correlation with longer amplicons and possible PCR bias towards unmethylated DNA for the intermediate methylation region (see Results and discussion ).

Article Snippet: Long-read SMRT-BS and reproducibility To assess if long-range PCR amplification introduces any bias based on methylation level, CpG islands with low (TUBGCP3 ), intermediate (MEST ), and high (EHPA8 ) methylation levels (previously determined by Illumina HumanMethylation450 BeadChip analysis) were subjected to SMRT-BS with four overlapping amplicons ranging from 625–1491 bp (Figure A; Additional file : Table S2).

Techniques: Methylation, Quantitation Assay, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing, Methylation Sequencing

Journal: NPJ Genomic Medicine

Article Title: Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet–Biedl Syndrome 9 (BBS9) deletion

doi: 10.1038/s41525-017-0042-3

Figure Lengend Snippet: Deletion breakpoint identification. The chromosome 7p14.3 genomic region is illustrated with tracks for the proband chromosomal microarray (CMA) results, genomic PCR mapping amplicon locations (1–9; green: amplified; red: did not amplify), and copy number variants detected among healthy individuals in the Database of Genomic Variants (DGV; blue: duplication; red: deletion) ( a ). Unambiguous breakpoint mapping was performed by long-read single molecule real-time (SMRT) sequencing (PacBio) of long-range PCR products that amplified across the deleted interval in the proband ( b ). These SMRT sequencing data were also aligned to a modified human genome reference that excluded the identified 72.8 kb deletion (chr7:33130616–33203409) ( c ), confirming that there were no other sequence alterations at the breakpoint locations. The precise deletion breakpoints were subsequently confirmed in the proband and both carrier parents by Sanger sequencing of the long-range PCR amplicons ( d )

Article Snippet: Long-read SMRT sequencing : Long-range PCR amplification across the identified breakpoint regions was accomplished using primers targeted to unique DNA sequences flanking the approximated deletion coordinates, and these amplicons were subjected to SMRTbell library construction and long-read SMRT sequencing (Pacific Biosciences, Menlo Park, CA).

Techniques: Microarray, Polymerase Chain Reaction, Amplification, Sequencing, Modification