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Solution hybrid selection capture for the recovery of functional full-length eukaryotic cDNAs from complex environmental samples.

Bragalini C, Ribière C, Parisot N, Vallon L, Prudent E, Peyretaillade E, Girlanda M, Peyret P, Marmeisse R, Luis P - DNA Res. (2014)

Bottom Line: After two successive rounds of capture, >90% of the resulting cDNAs were GH11 sequences, of which 70% (38 among 53 sequenced genes) were full length.Sequencing of polymerase chain reaction-amplified GH11 gene fragments from the captured sequences highlighted hundreds of phylogenetically diverse sequences that were not yet described, in public databases.This protocol offers the possibility of performing exhaustive exploration of eukaryotic gene families within microbial communities thriving in any type of environment.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Systems Biology, University of Turin, viale Mattioli 25, Turin 10125, Italy Ecologie Microbienne, UMR CNRS 5557, USC INRA 1364, Université de Lyon, Université Lyon 1, Villeurbanne 69622, France.

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Selectivity of the SHS capture. (A) Rank-abundance distribution of the most abundant GH11 nucleotide sequence clusters identified before (H0), or after one (H1) or two (H2) cycles of hybridization on the PUE cDNAs. Only clusters encompassing 80% of the sequences in the H0, H1 or H2 samples are shown. ‘C’ or ‘Y’ letters above bars indicate sequences obtained by random sequencing of plasmid inserts or which could be functionally expressed in yeast, respectively. (B) Venn diagram showing the number of unique or shared GH11 sequence clusters, before (H0), or after one (H1) or two (H2) cycles of hybridization on the PUE cDNAs. As in (A), only the most abundant clusters, encompassing 90% of the sequences, were used for the calculation. GH11 PCR sequences were clustered using a nucleotide sequence identity threshold of 95%. Similar Venn diagrams for the BRH, BRE and BEW samples are illustrated in Supplementary Fig. S5.
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DSU030F3: Selectivity of the SHS capture. (A) Rank-abundance distribution of the most abundant GH11 nucleotide sequence clusters identified before (H0), or after one (H1) or two (H2) cycles of hybridization on the PUE cDNAs. Only clusters encompassing 80% of the sequences in the H0, H1 or H2 samples are shown. ‘C’ or ‘Y’ letters above bars indicate sequences obtained by random sequencing of plasmid inserts or which could be functionally expressed in yeast, respectively. (B) Venn diagram showing the number of unique or shared GH11 sequence clusters, before (H0), or after one (H1) or two (H2) cycles of hybridization on the PUE cDNAs. As in (A), only the most abundant clusters, encompassing 90% of the sequences, were used for the calculation. GH11 PCR sequences were clustered using a nucleotide sequence identity threshold of 95%. Similar Venn diagrams for the BRH, BRE and BEW samples are illustrated in Supplementary Fig. S5.

Mentions: To evaluate the diversity of GH11 sequences at each step of the capture protocol, we performed a high-throughput Illumina MiSeq sequencing of GH11 amplicons obtained from all four cDNA samples, prior (H0) and after one (H1) or two (H2) cycles of SHS capture. Paired-end sequence reads were assembled to reconstitute the ca. 281-bp-long amplicons. Altogether, the total data set contained 334,161 full-length amplicon sequences that were clustered at a 95% nucleotide sequence identity threshold to produce a total number of 1,458 clusters, of which 1,001 (69%) were singletons (data summarized in Table 2 for each sample). Each of the 12 sequence data sets (4 cDNA samples × the 3 steps of the SHS) was characterized by few dominant clusters encompassing most of the sequences and a large number of clusters each containing a few, or even a single, sequences (illustrated in Fig. 3A for the PUE sample). None of the sequences obtained were identical to sequences deposited in databases. Only 17 of the sequence clusters, of which 14 exclusively from the BEW site, were >90% identical (maximum value of 97.5%) at the nucleotide level over their entire length to GH11 genes from either the Basidiomycota Tulasnella calospora or the Ascomycota Nectria haematococca and Pyrenophora teres.Table 2.


Solution hybrid selection capture for the recovery of functional full-length eukaryotic cDNAs from complex environmental samples.

Bragalini C, Ribière C, Parisot N, Vallon L, Prudent E, Peyretaillade E, Girlanda M, Peyret P, Marmeisse R, Luis P - DNA Res. (2014)

Selectivity of the SHS capture. (A) Rank-abundance distribution of the most abundant GH11 nucleotide sequence clusters identified before (H0), or after one (H1) or two (H2) cycles of hybridization on the PUE cDNAs. Only clusters encompassing 80% of the sequences in the H0, H1 or H2 samples are shown. ‘C’ or ‘Y’ letters above bars indicate sequences obtained by random sequencing of plasmid inserts or which could be functionally expressed in yeast, respectively. (B) Venn diagram showing the number of unique or shared GH11 sequence clusters, before (H0), or after one (H1) or two (H2) cycles of hybridization on the PUE cDNAs. As in (A), only the most abundant clusters, encompassing 90% of the sequences, were used for the calculation. GH11 PCR sequences were clustered using a nucleotide sequence identity threshold of 95%. Similar Venn diagrams for the BRH, BRE and BEW samples are illustrated in Supplementary Fig. S5.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263301&req=5

DSU030F3: Selectivity of the SHS capture. (A) Rank-abundance distribution of the most abundant GH11 nucleotide sequence clusters identified before (H0), or after one (H1) or two (H2) cycles of hybridization on the PUE cDNAs. Only clusters encompassing 80% of the sequences in the H0, H1 or H2 samples are shown. ‘C’ or ‘Y’ letters above bars indicate sequences obtained by random sequencing of plasmid inserts or which could be functionally expressed in yeast, respectively. (B) Venn diagram showing the number of unique or shared GH11 sequence clusters, before (H0), or after one (H1) or two (H2) cycles of hybridization on the PUE cDNAs. As in (A), only the most abundant clusters, encompassing 90% of the sequences, were used for the calculation. GH11 PCR sequences were clustered using a nucleotide sequence identity threshold of 95%. Similar Venn diagrams for the BRH, BRE and BEW samples are illustrated in Supplementary Fig. S5.
Mentions: To evaluate the diversity of GH11 sequences at each step of the capture protocol, we performed a high-throughput Illumina MiSeq sequencing of GH11 amplicons obtained from all four cDNA samples, prior (H0) and after one (H1) or two (H2) cycles of SHS capture. Paired-end sequence reads were assembled to reconstitute the ca. 281-bp-long amplicons. Altogether, the total data set contained 334,161 full-length amplicon sequences that were clustered at a 95% nucleotide sequence identity threshold to produce a total number of 1,458 clusters, of which 1,001 (69%) were singletons (data summarized in Table 2 for each sample). Each of the 12 sequence data sets (4 cDNA samples × the 3 steps of the SHS) was characterized by few dominant clusters encompassing most of the sequences and a large number of clusters each containing a few, or even a single, sequences (illustrated in Fig. 3A for the PUE sample). None of the sequences obtained were identical to sequences deposited in databases. Only 17 of the sequence clusters, of which 14 exclusively from the BEW site, were >90% identical (maximum value of 97.5%) at the nucleotide level over their entire length to GH11 genes from either the Basidiomycota Tulasnella calospora or the Ascomycota Nectria haematococca and Pyrenophora teres.Table 2.

Bottom Line: After two successive rounds of capture, >90% of the resulting cDNAs were GH11 sequences, of which 70% (38 among 53 sequenced genes) were full length.Sequencing of polymerase chain reaction-amplified GH11 gene fragments from the captured sequences highlighted hundreds of phylogenetically diverse sequences that were not yet described, in public databases.This protocol offers the possibility of performing exhaustive exploration of eukaryotic gene families within microbial communities thriving in any type of environment.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Systems Biology, University of Turin, viale Mattioli 25, Turin 10125, Italy Ecologie Microbienne, UMR CNRS 5557, USC INRA 1364, Université de Lyon, Université Lyon 1, Villeurbanne 69622, France.

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