Limits...
Utilization of super BAC pools and Fluidigm access array platform for high-throughput BAC clone identification: proof of concept.

Maughan PJ, Smith SM, Raney JA - J. Biomed. Biotechnol. (2012)

Bottom Line: Forty-six percent of the SNPs were associated with a single unambiguous identified BAC clone.PCR amplification and next-generation sequencing of individual BAC clones confirmed the IFC clone identification.Utilization of the Fluidigm Dynamic array platform allowed for the simultaneous PCR screening of 10,752 BAC pools for 96 SNP tag sites in less than three hours at a cost of ~$0.05 per reaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant & Wildlife Sciences, Brigham Young University, Provo, UT 84602, USA. jeff maughan@byu.edu

ABSTRACT
Bacterial artificial chromosome (BAC) libraries are critical for identifying full-length genomic sequences, correlating genetic and physical maps, and comparative genomics. Here we describe the utilization of the Fluidigm access array genotyping system in conjunction with KASPar genotyping technology to identify individual BAC clones corresponding to specific single-nucleotide polymorphisms (SNPs) from an Amplicon Express seven-plate super pooled Amaranthus hypochondriacus BAC library. Ninety-six SNP loci, spanning the length of A. hypochondriacus linkage groups 1, 2, and 15, were simultaneously tested for clone identification from four BAC super pools, corresponding to 28 384-well plates, using a single Fluidigm integrated fluidic chip (IFC). Forty-six percent of the SNPs were associated with a single unambiguous identified BAC clone. PCR amplification and next-generation sequencing of individual BAC clones confirmed the IFC clone identification. Utilization of the Fluidigm Dynamic array platform allowed for the simultaneous PCR screening of 10,752 BAC pools for 96 SNP tag sites in less than three hours at a cost of ~$0.05 per reaction.

Show MeSH
BAC pool screening using the Fluidigm Access Array. (a) shows assay AM25002 which was detected at a single BAC library address, specifically in Plate 1, row O, column 5 of Super Pool x no. 3 (SP3-1O5). (b) shows assay AM24655 which was detected at two library addresses in Super Pools no. 2 (SP2-2L18) and no. 4 (SP4-7K16). No template controls (NTC) are identified at the origin of each Cartesian graph. Genomic DNA of the amaranth cultivar “Plainsman” was used as positive control samples. Successfully amplified pooled BAC clones are circled with a dashed line and are identified as being either Column (C-), Row (R-) or Plate (P-) pools followed by the specific columns, rows or plates utilized in those pools. Pools without the target sequence fail to amplify and are found near the NTC.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3403795&req=5

fig3: BAC pool screening using the Fluidigm Access Array. (a) shows assay AM25002 which was detected at a single BAC library address, specifically in Plate 1, row O, column 5 of Super Pool x no. 3 (SP3-1O5). (b) shows assay AM24655 which was detected at two library addresses in Super Pools no. 2 (SP2-2L18) and no. 4 (SP4-7K16). No template controls (NTC) are identified at the origin of each Cartesian graph. Genomic DNA of the amaranth cultivar “Plainsman” was used as positive control samples. Successfully amplified pooled BAC clones are circled with a dashed line and are identified as being either Column (C-), Row (R-) or Plate (P-) pools followed by the specific columns, rows or plates utilized in those pools. Pools without the target sequence fail to amplify and are found near the NTC.

Mentions: The high-throughput screening of the pooled library employed a Fluidigm 96.96 Dynamic Array genotyping platform using KASPar genotyping chemistry. The Fluidigm platform uses an integrated fluidic chip to create 9,216 9.6 nL PCR reactions. Thus four super pool libraries, each consisting of 23 pooled samples (Figure 1), could be screened simultaneously with 96 STS targets. We also included two positive controls (genomic DNA from the cultivar “Plainsman”) and two no template controls (NTCs). Setup of the IFC can be accomplished with an 8-channel multichannel pipettor in less than 1 hour. The use of the fluorescence KASPar genotyping chemistry eliminates the need for detection of the amplified PCR product using radiography or electrophoresis, since successful amplification is detected by a fluorescent signal. Thus in our proof-of-concept experiment we screened 10,752 BAC clones, arrayed in four super pools, with 96 SNP assays using a single IFC. The SNP assays utilized represented SNP loci that were distributed across three linkage groups of a recently published A. hypochondriacus linkage map, specifically linkage groups 1, 2, and 15 (Figure 2). The complete list of SNP markers utilized, along with their GenBank accession number, SNP type, and primer sequences can be found in Supplemental Table S1 in Supplementary Material available online at doi: 10.1155/2012/405940. Of the 96 SNP targets, 5 (5.2%) failed to amplify in any of the BAC DNA pools or with the positive genomic DNA control, while 23 (23.9%) amplified in only the positive genomic DNA control sample (no amplification in the BAC pools). This was somewhat surprising since a 3.4X library should have approximately 92% representation of all the genome sequences, suggesting that the reported genome size of A. hypochondriacus may be slightly larger than originally reported (466 Mb/C; Bennett and Smith [26]) or that the BAC library, developed through partial HindIII digestion, may be underrepresented in some genomic regions. Sixty-eight (70.8%) of the clones amplified in one or more of the BAC pool samples, of which 44 (64.7%) could be unambiguously assigned to specific BAC clone library addresses. The remaining 24 (35.3%) could not be assigned to an unambiguous BAC clone address, since two or more positive clones were present in a single super pool. We note that while specific BAC clone addresses could not be identified for these 24 SNP assays, specific plates, and often, specific columns and/or rows could be determined. Representative images of the BAC pool screening results are shown in Figure 3.


Utilization of super BAC pools and Fluidigm access array platform for high-throughput BAC clone identification: proof of concept.

Maughan PJ, Smith SM, Raney JA - J. Biomed. Biotechnol. (2012)

BAC pool screening using the Fluidigm Access Array. (a) shows assay AM25002 which was detected at a single BAC library address, specifically in Plate 1, row O, column 5 of Super Pool x no. 3 (SP3-1O5). (b) shows assay AM24655 which was detected at two library addresses in Super Pools no. 2 (SP2-2L18) and no. 4 (SP4-7K16). No template controls (NTC) are identified at the origin of each Cartesian graph. Genomic DNA of the amaranth cultivar “Plainsman” was used as positive control samples. Successfully amplified pooled BAC clones are circled with a dashed line and are identified as being either Column (C-), Row (R-) or Plate (P-) pools followed by the specific columns, rows or plates utilized in those pools. Pools without the target sequence fail to amplify and are found near the NTC.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3403795&req=5

fig3: BAC pool screening using the Fluidigm Access Array. (a) shows assay AM25002 which was detected at a single BAC library address, specifically in Plate 1, row O, column 5 of Super Pool x no. 3 (SP3-1O5). (b) shows assay AM24655 which was detected at two library addresses in Super Pools no. 2 (SP2-2L18) and no. 4 (SP4-7K16). No template controls (NTC) are identified at the origin of each Cartesian graph. Genomic DNA of the amaranth cultivar “Plainsman” was used as positive control samples. Successfully amplified pooled BAC clones are circled with a dashed line and are identified as being either Column (C-), Row (R-) or Plate (P-) pools followed by the specific columns, rows or plates utilized in those pools. Pools without the target sequence fail to amplify and are found near the NTC.
Mentions: The high-throughput screening of the pooled library employed a Fluidigm 96.96 Dynamic Array genotyping platform using KASPar genotyping chemistry. The Fluidigm platform uses an integrated fluidic chip to create 9,216 9.6 nL PCR reactions. Thus four super pool libraries, each consisting of 23 pooled samples (Figure 1), could be screened simultaneously with 96 STS targets. We also included two positive controls (genomic DNA from the cultivar “Plainsman”) and two no template controls (NTCs). Setup of the IFC can be accomplished with an 8-channel multichannel pipettor in less than 1 hour. The use of the fluorescence KASPar genotyping chemistry eliminates the need for detection of the amplified PCR product using radiography or electrophoresis, since successful amplification is detected by a fluorescent signal. Thus in our proof-of-concept experiment we screened 10,752 BAC clones, arrayed in four super pools, with 96 SNP assays using a single IFC. The SNP assays utilized represented SNP loci that were distributed across three linkage groups of a recently published A. hypochondriacus linkage map, specifically linkage groups 1, 2, and 15 (Figure 2). The complete list of SNP markers utilized, along with their GenBank accession number, SNP type, and primer sequences can be found in Supplemental Table S1 in Supplementary Material available online at doi: 10.1155/2012/405940. Of the 96 SNP targets, 5 (5.2%) failed to amplify in any of the BAC DNA pools or with the positive genomic DNA control, while 23 (23.9%) amplified in only the positive genomic DNA control sample (no amplification in the BAC pools). This was somewhat surprising since a 3.4X library should have approximately 92% representation of all the genome sequences, suggesting that the reported genome size of A. hypochondriacus may be slightly larger than originally reported (466 Mb/C; Bennett and Smith [26]) or that the BAC library, developed through partial HindIII digestion, may be underrepresented in some genomic regions. Sixty-eight (70.8%) of the clones amplified in one or more of the BAC pool samples, of which 44 (64.7%) could be unambiguously assigned to specific BAC clone library addresses. The remaining 24 (35.3%) could not be assigned to an unambiguous BAC clone address, since two or more positive clones were present in a single super pool. We note that while specific BAC clone addresses could not be identified for these 24 SNP assays, specific plates, and often, specific columns and/or rows could be determined. Representative images of the BAC pool screening results are shown in Figure 3.

Bottom Line: Forty-six percent of the SNPs were associated with a single unambiguous identified BAC clone.PCR amplification and next-generation sequencing of individual BAC clones confirmed the IFC clone identification.Utilization of the Fluidigm Dynamic array platform allowed for the simultaneous PCR screening of 10,752 BAC pools for 96 SNP tag sites in less than three hours at a cost of ~$0.05 per reaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant & Wildlife Sciences, Brigham Young University, Provo, UT 84602, USA. jeff maughan@byu.edu

ABSTRACT
Bacterial artificial chromosome (BAC) libraries are critical for identifying full-length genomic sequences, correlating genetic and physical maps, and comparative genomics. Here we describe the utilization of the Fluidigm access array genotyping system in conjunction with KASPar genotyping technology to identify individual BAC clones corresponding to specific single-nucleotide polymorphisms (SNPs) from an Amplicon Express seven-plate super pooled Amaranthus hypochondriacus BAC library. Ninety-six SNP loci, spanning the length of A. hypochondriacus linkage groups 1, 2, and 15, were simultaneously tested for clone identification from four BAC super pools, corresponding to 28 384-well plates, using a single Fluidigm integrated fluidic chip (IFC). Forty-six percent of the SNPs were associated with a single unambiguous identified BAC clone. PCR amplification and next-generation sequencing of individual BAC clones confirmed the IFC clone identification. Utilization of the Fluidigm Dynamic array platform allowed for the simultaneous PCR screening of 10,752 BAC pools for 96 SNP tag sites in less than three hours at a cost of ~$0.05 per reaction.

Show MeSH