 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Sep 09, 2024 |
| Title |
Bisulfite converted 5'-3' strand gDNA mCpG |
| Sample type |
SRA |
| |
|
| Source name |
HeLa human genomic DNA
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: HeLa human genomic DNA
cell line: HeLa
cell type: adenocarcinoma
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Reaction mixtures for linear PCR contained 10 µM dGTP and 200 µM d(A/T/C)TP (each), 100 nM 109 bp forward primer, template DNA (3 fM 803 bp C or 5mC template generated by PCR, 62.5 ng HeLa gDNA native or CpG methylated (New England Biolabs)) and 300 nM RIV A8 KTq variant in 1x KTq reaction buffer (50 mM Tris HCl (pH 9.2), 16 mM (NH4)2SO4, 2.5 mM MgCl2, 0.1% (v/v) Tween 20) in 25 µL end volume. PCR was performed with an initial denaturation at 95°C for 3 min followed by amplification over 20 cycles with denaturation at 95°C for 10 s, annealing at 60°C for 30 s and elongation at 72°C for 10 min. Reaction mixtures were purified by using the NucleoSpin® Gel and PCR Clean-up XS kit (Macherey-Nagel) in combination with the NTC binding buffer according to the manufacturer’s instructions for ssDNA PCR product purification. DNA was eluted with 18 µL Elution Buffer NE (5 mM Tris HCl (pH 8.5)). To use the linear PCR product as template for NGS library preparation, the ssDNA was exponentially amplified by a high-fidelity DNA polymerase to increase the 109 bp product concentration. Reaction mixtures contained 200 µM dNTPs (each), 100 nM 109 bp forward and reverse primer, 67% (v/v) of the PCR elution and 0.02 U/µL Q5® Hot Start High-Fidelity DNA Polymerase in 1x Q5® Reaction Buffer (New England Biolabs). PCR was performed in 25 µL reaction mixtures with an initial denaturation at 98°C for 30 s followed by amplification over 10 cycles with denaturation at 98°C for 5 s, annealing at 62°C for 10 s and elongation at 72°C for 5 s. Reaction mixtures were purified by using the NucleoSpin® Gel and PCR Clean-up XS kit (Macherey-Nagel) and a 1:2 dilution of the NTI buffer according to the manufacturer’s instructions to optimise PCR product purification and yield. DNA was eluted with 18 µL Elution Buffer NE. Note, all reactions were performed in the Thermo Scientific™ Low Profile Tubes (Thermo Scientific) and DNA was eluted in Eppendorf DNA LoBind® Tubes (Eppendorf). 16.25 µL of purified linear PCR product dsDNA was applied as template for a 2-cycle UMI PCR. Reaction mixtures for UMI introduction contained 200 µM dNTPs (each), 200 nM forward and reverse UMI primer, 0.02 U/µL Q5® Hot Start High-Fidelity DNA Polymerase and 1x Q5® Reaction Buffer (New England Biolabs) in 25 µL end volume. PCR was performed with an initial denaturation at 98°C for 30 s followed by primer elongation over 2 cycles with denaturation at 98°C for 10 s, annealing at 48°C for 30 s and elongation at 72°C for 30 s. Final elongation was performed for 2 min at 72°C. 183 bp UMI PCR product was purified by preparative agarose gel electrophoresis using the NucleoSpin® Gel and PCR Clean-up XS kit (Macherey-Nagel) according to the manufacturer’s instructions. Purified UMI PCR product was eluted with 18 µL Elution Buffer NE. DNA concentration was determined by qPCR according to the method described for NGS library preparation using template DNA generated by PCR. DNA repair using the PreCR® Repair Mix (New England Biolabs) and Amplicon PCR were performed according to the method described for NGS library preparation using template DNA generated by PCR. Amplicon PCR was performed with an initial denaturation at 98°C for 30 s followed by amplification over 30 cycles with denaturation at 98°C for 10 s, annealing at 70°C for 30 s and elongation at 72°C for 30 s. Final elongation was performed for 2 min at 72°C. 263 bp Amplicon PCR product was purified by preparative agarose gel electrophoresis using the NucleoSpin® Gel and PCR Clean-up XS kit (Macherey-Nagel) according to the manufacturer’s instructions. To improve purification, non-diluted NTI binding buffer was used first for solubilising agarose gel slices and subsequently the solved mixture was diluted in total to 1:6.5 with Milli-Q water prior to DNA binding on the purification column. PCR product was eluted with Elution Buffer NE. Treatment of extracted DNA with PreCR® Repair Mix (New England Biolabs) and QIAEX II system (Qiagen) purification were performed according to the method described for NGS library preparation using template DNA generated by PCR. Repaired product DNA was eluted with 22 µL Milli-Q water. DNA library concentration determination, quality control and sequencing was performed as described for NGS library preparation using template DNA generated by PCR. Note, all reactions were performed in the Thermo Scientific™ Low Profile Tubes (Thermo Scientific) and DNA was eluted in Eppendorf DNA LoBind® Tubes (Eppendorf).
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
| |
|
| Description |
gDNA mCpG
|
| Data processing |
Sequencing data were processed by using the open source software KNIME 4.6.2
Raw sequence and quality values were extracted from the FASTQ file format and Phred quality scores (Q scores) were transformed into base calling error probabilities (P)
The data was preprocessed by defining the unique molecular identifier (UMI) sequence context and translating Read 1 sequence and P into the reverse complement orientation
Read 1 and Read 2 were aligned to give the expected size of the template (bisulfite converted samples 95 bp, 5mC detection samples 109 bp) and merged into one sequence for which base calls with the lower error probability were transferred for the Read 1 and Read 2 overlay segment
High quality data was filtered by removal of reads containing a base within the UMI contexts with a P value above the threshold or removal of merged reads with a mean error probability over all bases in the sequence context above the threshold value
For each filtered read, a deletion and insertion correction was performed to adjust frameshifts by employing the Levenshtein distance between the read and reference template (OR10A2 olfactory receptor family 10 subfamily A member 2 [ Homo sapiens (human) ])
N base calls were replaced by reference bases to prevent false positive error detection
Reads were aligned to the reference template sequence and reads with a misalignment higher as 6% or 12% were removed from the data set
Reads were sorted into UMI family groups with identical UMIs and analysis was proceeded with UMI families containing a minimum of three reads
Error calculation of UMI families was performed by averaging over the error for each sequence position of each read in one UMI family
By employing an error cut-off of 0.9, true KlenTaq DNA polymerase derived errors were then set to 1 and errors below the cut-off were set to 0 for each UMI family
The mean error was calculated over all UMI families at each sequence position, yielding the KTq based error rate
5mC detection was facilitated by comparing the error rates of the unmodified C template data set with the error rates of the 5mC template sequencing data using the Microsoft Excel software
Error rates, calculated from sequencing data of human genomic DNA based NGS libraries; were processed further for 5mC detection using the Microsoft Excel software. Errors were standardised using an adapted z-score which only includes error rates at C positions but values from CpG sites were excluded
Assembly: OR10A2 olfactory receptor family 10 subfamily A member 2 [ >NC_000011.10 Homo sapiens chromosome 11, GRCh38.p14 Primary Assembly ]
Supplementary files format and content: excel-files include position, reference DNA sequence and error rates
Library strategy: DNA Amplicon-Seq
|
| |
|
| Submission date |
May 26, 2023 |
| Last update date |
Sep 09, 2024 |
| Contact name |
Melanie Henkel |
| E-mail(s) |
melanie.henkel@uni-konstanz.de
|
| Organization name |
University of Konstanz
|
| Department |
Department of Chemistry
|
| Lab |
AG Marx
|
| Street address |
Universitätsstraße 10
|
| City |
Konstanz |
| State/province |
Baden-Württemberg |
| ZIP/Postal code |
78464 |
| Country |
Germany |
| |
|
| Platform ID |
GPL30173 |
| Series (1) |
| GSE233599 |
Detection of 5mC in DNA templates generated by PCR and HeLa human genomic DNA by linear PCR using KTq DNA polymerase variants |
|
| Relations |
| BioSample |
SAMN35440908 |
| SRA |
SRX20526603 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7431035_AM165_Al12_Min3.xlsx |
17.8 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
