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Next generation sequencing in paleomicrobiology
Abstracts / New Biotechnology 33S (2016) S1–S213
In order to address this problem, we have been developing a novel CRISPR-nickase system. In this system, we used Cas9 nickase instead of wild-type Cas9, which induces a single strand break in the gRNA targeting DNA. Since the single strand breaks are repaired through HDR or a simple gap filling pathway, unnecessary mutations were not introduced through NHEJ, when the target and PAM sequences still remained after nickase-mediated HDR. This system was able to efficiently and precisely edit bases in the target sequence, PAM sequence, and even 50 bp-upstream bases from PAM, indicating that our CRISPR-nickase system can cover almost all bases in the yeast genome [1]. Reference [1] A. Satomura, et al. submitted.
http://dx.doi.org/10.1016/j.nbt.2016.06.947
O23-5 Next generation sequencing in paleomicrobiology Dominik Strapagiel 1,∗ , Paulina Borówka 1 , Wiesław Lorkiewicz 1 , 1 , Anna Brzostek 2 , Jarosław Dziadek 3 , ´ Beata Borowska-Struginska 2 ˙ ˙ ˛ ´ Elzbieta Zadzi nska 1
University of Lodz, Poland Institute for Medical Biology, Poland 3 Polish Academy of Sciences, Poland 2
The aim of the study was to apply NGS technique to detect the incidence of tuberculosis in the Neolithic populations from Kujawy. 3/30 skeletons show lesions which are suggestive of skeletal TB. PCR assays aimed at detecting IS6110 repeat elements proved positive results for another two individuals. However, the specificity of IS6110 has been recently questioned because identical or similar elements are present in mycobacteria other than tuberculosis (MOTT). Among these may be soil-inhabiting mycobacterial species which can populate archaeological skeletons, so sequencing using NGS of other genomic regions is necessary for confirmation of the presence of MTBC. A reference sequence was constructed from M. tuberculosis genome, which was filtered out with BLAST tool against MOTT. Finally we have obtained the target reference sequence with 1536 genes which are crucial for MTBC detection (total length of 816 kbp). We have calculated index of number of reads matching to reference/total number of non-human reads. Only one individual (PCR negative) gave strong signal of MTBC and was indicated as the outlier (more than +3SD from medium index). The lack of a concordance between results obtained by the morphological examination, PCRs assays and NGS analysis is not surprising taking into account that: (1) there are no pathognomonic skeletal indicators of TB; (2) bone changes occur only in about 3–7% of individuals with active TB; (3) taphonomic degradation of a DNA. The use of complementary methods based on massive parallel sequencing allows more accurate assessment of prevalence of TB in past human populations. http://dx.doi.org/10.1016/j.nbt.2016.06.948
S65
O23-6 Antisense oligomers as therapeutic agents neutralizing toxic RNA in myotonic dystrophy Krzysztof Sobczak Adam Mickiewicz University, Poland Myotonic dystrophy type 1 (DM1) is an RNA dominant disorder caused by an expansion of a CTG repeat in DMPK gene. Typical clinical features of DM1 are associated with myotonia, atrophy and weakness of skeletal muscles. The mutated DMPK transcripts containing toxic, expanded CUG repeats (CUGexp ) accumulate in a nuclear foci and sequester some nuclear proteins regulating RNA metabolism, mostly MBNLs. The sequestration of MBNLs in muscles of DM patients and mouse models impact on global abnormalities in alternative splicing of hundreds of mRNAs. We decided to test several strategies to eliminate or reduce the toxic effect of CUGexp in different DM models. They include the siRNA-induced degradation of CUGexp and inhibition of nuclear protein sequestration by antisense oligomers which specifically bind to CUGexp . The in vitro and in vivo studies showed that different types of chemically modified antisense oligonucleotides (AONs) composed of CAG repeat sequence bound to CUGexp RNA with high affinity and prevent formation of CUGexp /MBNL interaction. On the other hand, siRNA designed to interact with CUG repeats induces efficient silencing of CUGexp transcript in vivo. The DM1-patients derived fibroblasts and skeletal muscles of DM mouse model treated with these AONs and siRNAs showed (i) reduction of the number and size of CUGexp foci, (ii) correction of MBNL-dependent alternative splicing caused by reduction of protein sequestration and (iii) significant reduction of myotonia. The beneficial effects were observed in vivo for relatively long time. Our data showed that short AONs and siRNA are potential therapeutic agent in DM1 treatment. http://dx.doi.org/10.1016/j.nbt.2016.06.949
O23-7 Not just PCR: using modified oligonucleotides for recursive cloning Bob Van Hove ∗ , Chiara Guidi, Lien De Wannemaeker, Jo Maertens, Marjan De Mey Ghent University, Belgium Molecular cloning has come a long way since the 1970s, and today’s DNA assembly techniques boast much improved efficiency, accuracy and versatility. However, an issue rarely tackled by modern methods arises after the assembly reaction: How can additional parts be introduced into the construct without running the risk of PCR-derived mutations in the already assembled sequence? In this view, we have developed a novel Golden Gate cloning derivative that enables iterative cloning using only a single restriction enzyme, without relying on PCR. The basic principle behind our technique is that, together with the part to be cloned, a new cloning site is introduced as well. To prevent this site from being destroyed itself during the one-pot restriction and ligation reaction, it is supplied in the form of methylated oligonucleotides that are not recognised by the restriction enzyme. Replication in E. coli removes the methylation pattern, resulting in an assembled construct that can receive additional parts. This approach, which we have called Protected Oligonucleotide Duplex Assisted Cloning (PODAC), enables a recursive cloning workflow because the same restriction enzyme is used at each iter-
In order to address this problem, we have been developing a novel CRISPR-nickase system. In this system, we used Cas9 nickase instead of wild-type Cas9, which induces a single strand break in the gRNA targeting DNA. Since the single strand breaks are repaired through HDR or a simple gap filling pathway, unnecessary mutations were not introduced through NHEJ, when the target and PAM sequences still remained after nickase-mediated HDR. This system was able to efficiently and precisely edit bases in the target sequence, PAM sequence, and even 50 bp-upstream bases from PAM, indicating that our CRISPR-nickase system can cover almost all bases in the yeast genome [1]. Reference [1] A. Satomura, et al. submitted.
http://dx.doi.org/10.1016/j.nbt.2016.06.947
O23-5 Next generation sequencing in paleomicrobiology Dominik Strapagiel 1,∗ , Paulina Borówka 1 , Wiesław Lorkiewicz 1 , 1 , Anna Brzostek 2 , Jarosław Dziadek 3 , ´ Beata Borowska-Struginska 2 ˙ ˙ ˛ ´ Elzbieta Zadzi nska 1
University of Lodz, Poland Institute for Medical Biology, Poland 3 Polish Academy of Sciences, Poland 2
The aim of the study was to apply NGS technique to detect the incidence of tuberculosis in the Neolithic populations from Kujawy. 3/30 skeletons show lesions which are suggestive of skeletal TB. PCR assays aimed at detecting IS6110 repeat elements proved positive results for another two individuals. However, the specificity of IS6110 has been recently questioned because identical or similar elements are present in mycobacteria other than tuberculosis (MOTT). Among these may be soil-inhabiting mycobacterial species which can populate archaeological skeletons, so sequencing using NGS of other genomic regions is necessary for confirmation of the presence of MTBC. A reference sequence was constructed from M. tuberculosis genome, which was filtered out with BLAST tool against MOTT. Finally we have obtained the target reference sequence with 1536 genes which are crucial for MTBC detection (total length of 816 kbp). We have calculated index of number of reads matching to reference/total number of non-human reads. Only one individual (PCR negative) gave strong signal of MTBC and was indicated as the outlier (more than +3SD from medium index). The lack of a concordance between results obtained by the morphological examination, PCRs assays and NGS analysis is not surprising taking into account that: (1) there are no pathognomonic skeletal indicators of TB; (2) bone changes occur only in about 3–7% of individuals with active TB; (3) taphonomic degradation of a DNA. The use of complementary methods based on massive parallel sequencing allows more accurate assessment of prevalence of TB in past human populations. http://dx.doi.org/10.1016/j.nbt.2016.06.948
S65
O23-6 Antisense oligomers as therapeutic agents neutralizing toxic RNA in myotonic dystrophy Krzysztof Sobczak Adam Mickiewicz University, Poland Myotonic dystrophy type 1 (DM1) is an RNA dominant disorder caused by an expansion of a CTG repeat in DMPK gene. Typical clinical features of DM1 are associated with myotonia, atrophy and weakness of skeletal muscles. The mutated DMPK transcripts containing toxic, expanded CUG repeats (CUGexp ) accumulate in a nuclear foci and sequester some nuclear proteins regulating RNA metabolism, mostly MBNLs. The sequestration of MBNLs in muscles of DM patients and mouse models impact on global abnormalities in alternative splicing of hundreds of mRNAs. We decided to test several strategies to eliminate or reduce the toxic effect of CUGexp in different DM models. They include the siRNA-induced degradation of CUGexp and inhibition of nuclear protein sequestration by antisense oligomers which specifically bind to CUGexp . The in vitro and in vivo studies showed that different types of chemically modified antisense oligonucleotides (AONs) composed of CAG repeat sequence bound to CUGexp RNA with high affinity and prevent formation of CUGexp /MBNL interaction. On the other hand, siRNA designed to interact with CUG repeats induces efficient silencing of CUGexp transcript in vivo. The DM1-patients derived fibroblasts and skeletal muscles of DM mouse model treated with these AONs and siRNAs showed (i) reduction of the number and size of CUGexp foci, (ii) correction of MBNL-dependent alternative splicing caused by reduction of protein sequestration and (iii) significant reduction of myotonia. The beneficial effects were observed in vivo for relatively long time. Our data showed that short AONs and siRNA are potential therapeutic agent in DM1 treatment. http://dx.doi.org/10.1016/j.nbt.2016.06.949
O23-7 Not just PCR: using modified oligonucleotides for recursive cloning Bob Van Hove ∗ , Chiara Guidi, Lien De Wannemaeker, Jo Maertens, Marjan De Mey Ghent University, Belgium Molecular cloning has come a long way since the 1970s, and today’s DNA assembly techniques boast much improved efficiency, accuracy and versatility. However, an issue rarely tackled by modern methods arises after the assembly reaction: How can additional parts be introduced into the construct without running the risk of PCR-derived mutations in the already assembled sequence? In this view, we have developed a novel Golden Gate cloning derivative that enables iterative cloning using only a single restriction enzyme, without relying on PCR. The basic principle behind our technique is that, together with the part to be cloned, a new cloning site is introduced as well. To prevent this site from being destroyed itself during the one-pot restriction and ligation reaction, it is supplied in the form of methylated oligonucleotides that are not recognised by the restriction enzyme. Replication in E. coli removes the methylation pattern, resulting in an assembled construct that can receive additional parts. This approach, which we have called Protected Oligonucleotide Duplex Assisted Cloning (PODAC), enables a recursive cloning workflow because the same restriction enzyme is used at each iter-