The authors would like to thank the laboratory staff of the Max Planck Institute for the Science of Human History for their help in the development and implementation of these protocols. This work would not have been possible without the input and hard work of Dr. Guido Brandt, Dr. Elizabeth Nelson, Antje Wissegot, and Franziska Aron. This study was funded by the Max Planck Society, the European Research Council (ERC) under the European Union&#39;s Horizon 2020 research and innovation program under grant agreements No 771234 - PALEoRIDER (WH, ABR) and Starting Grant No. 805268 CoDisEASe (to KIB).

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H., Bl&#228;uer, A., Rannam&#228;e, E., Boessenkool, S., Hallsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Not+a+limitless+resource:+ethics+and+guidelines+for+destructive+sampling+of+archaeofaunal+remains.">Not a limitless resource: ethics and guidelines for destructive sampling of archaeofaunal remains.</a> Royal Society Open Science. 6 (10), 191059 (2019).</li><li>Pinhasi, R., Fernandes, D. M., Sirak, K., Cheronet, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating+the+human+cochlea+to+generate+bone+powder+for+ancient+DNA+analysis.">Isolating the human cochlea to generate bone powder for ancient DNA analysis.</a> Nature Protocols. 14 (4), 1194-1205 (2019).</li><li>Latham, K. E., Miller, J. 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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+ancient+DNA+preservation+in+petrous+bone+and+tooth+cementum.">Comparing ancient DNA preservation in petrous bone and tooth cementum.</a> PLoS ONE. 12 (1), 0170940 (2017).</li><li>Sirak, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+auditory+ossicles+as+an+alternative+optimal+source+of+ancient+DNA.">Human auditory ossicles as an alternative optimal source of ancient DNA.</a> Genome Research. 30 (3), 427-436 (2020).</li><li>Parker, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+investigation+of+human+DNA+preservation+in+medieval+skeletons.">A systematic investigation of human DNA preservation in medieval skeletons.</a> Scientific Reports. 10 (1), 18225 (2020).</li><li>Pinhasi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+ancient+DNA+yields+from+the+inner+ear+part+of+the+human+petrous+bone.">Optimal ancient DNA yields from the inner ear part of the human petrous bone.</a> PLoS ONE. 10 (6), 0129102 (2015).</li><li>Sirak, K. 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The authors have no conflicts of interest to report.

Current practice in ancient human population genetics is to preferentially sample from the pars petrosa (step 2.1) whenever possible. However, the pars petrosa can be a difficult sample to obtain, as it is highly valued for a myriad of skeletal assessments (e.g., population history32, the estimation of fetal age at death33, and sex determination34), and, historically, sampling of the pars petrosa for DNA analysis can be highly destructive3,4 (including the protocol presented here, although new, minimally invasive protocols13,14 have now been widely adopted to alleviate this concern). This is compounded by the fact that, until very recently, a large-scale, systematic study of human DNA recovery across the skeleton had not been attempted11, making finding an appropriate sampling strategy when the petrous pyramid is unavailable challenging.
The protocols presented here help to alleviate that challenge by providing a set of optimized procedures for DNA sampling from archaeological/forensic skeletal remains including the pars petrosa as well as seven alternate anatomical sampling locations across four additional skeletal elements. The critical steps included are all intended to minimize the possibility of DNA loss/damage due to either inefficient sampling (steps 2.1.6 and 3.2.1.3) or overheating of samples during drilling/cutting (step 3.1.6). Additionally, it has been noted throughout the protocol that it may be necessary to modify/omit the pre-treatment steps to ensure the best performance in highly degraded samples. It should also be noted that even among the selected elements presented here, there remain several possible alternative sampling techniques (particularly for the pars petrosa13,14), as well as ample room for further optimization of the underexploited anatomical sampling locations presented here (i.e.,&#160;the talus: step 2.5 and the vertebrae: step 2.3).
It is also important to keep in mind that these protocols have been designed and tested using ancient juvenile-adult remains of high quality (good morphological preservation) for the purposes of endogenous human DNA analyses. The results presented may not extend to more highly degraded materials, other preservation contexts, infant remains, non-human remains, or studies of pathogens or the microbiome, as a greater exploration into the use of these protocols in additional contexts is still needed. Additionally, the alternative skeletal elements presented here (the teeth, vertebrae, distal phalanx, and tali) may be challenging to assign to a single individual among commingled remains, necessitating sampling from multiple elements to ensure a single origin. Despite these limitations, making these protocols widely available can help alleviate some of the heterogeneity surrounding sample selection and processing by providing a generalized and quantitatively optimized framework for use in a wide range of future aDNA/forensic studies on human remains.

The sampling of ancient human remains for the purposes of DNA recovery and analysis is inherently destructive1,2,3,4. The samples themselves are precious specimens and morphological preservation should be preserved wherever possible. As such, it is imperative that sampling practices be optimized to both avoid unnecessary destruction of irreplaceable material and to maximize the probability of success. Current best practice techniques are based on a small cohort of studies limited to either forensic surveys5,6, studies of ancient specimens where the development of optimal sampling is not the direct aim of the study7, or dedicated studies utilizing either non-human remains8 or targeting a very small selection of anatomical sampling locations (used here to denote a specific area of a skeletal element from which bone powder, for use in downstream DNA analyses, was generated)9,10. The sampling protocols presented here were optimized in the first large-scale systematic study of DNA preservation across multiple skeletal elements from multiple individuals11. All samples stemmed from skeletal elements recovered from 11 individuals excavated from the church graveyard of the abandoned medieval settlement of Krakauer Berg near Pei&#223;en, Saxony-Anhalt, Germany (see Table 1 for detailed sample demographics) and, as such, may need modification for use with samples outside of this geographical/temporal range.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Individual</td>
			<td>Sex</td>
			<td>Estimated age at death</td>
			<td>14C dates (CE, Cal 2-sigma)</td>
		</tr>
		<tr>
			<td>KRA001</td>
			<td>Male</td>
			<td>25-35</td>
			<td>1058-1219</td>
		</tr>
		<tr>
			<td>KRA002</td>
			<td>Female</td>
			<td>20-22</td>
			<td>1227-1283</td>
		</tr>
		<tr>
			<td>KRA003</td>
			<td>Male</td>
			<td>25</td>
			<td>1059-1223</td>
		</tr>
		<tr>
			<td>KRA004</td>
			<td>Male</td>
			<td>15</td>
			<td>1284-1392</td>
		</tr>
		<tr>
			<td>KRA005</td>
			<td>Male</td>
			<td>10-12</td>
			<td>1170-1258</td>
		</tr>
		<tr>
			<td>KRA006</td>
			<td>Female</td>
			<td>30-40</td>
			<td>1218-1266</td>
		</tr>
		<tr>
			<td>KRA007</td>
			<td>Female</td>
			<td>25-30</td>
			<td>1167-1251</td>
		</tr>
		<tr>
			<td>KRA008</td>
			<td>Male</td>
			<td>20</td>
			<td>1301-1402</td>
		</tr>
		<tr>
			<td>KRA009</td>
			<td>Male</td>
			<td>Unknown</td>
			<td>1158-1254</td>
		</tr>
		<tr>
			<td>KRA010</td>
			<td>Male</td>
			<td>25</td>
			<td>1276-1383</td>
		</tr>
		<tr>
			<td>KRA011</td>
			<td>Female</td>
			<td>30-45</td>
			<td>1040-1159</td>
		</tr>
	</tbody>
</table>
Table 1: Genetically determined sex, archaeologically determined estimated age at death, and radiocarbon dating (14C Cal 2-sigma) for all the 11 individuals sampled. This table has been adapted from Parker, C. et al. 202011.
These protocols allow for a relatively straightforward and efficient generation of bone powder from eight anatomical sampling locations across five skeletal elements (including the pars petrosa) with limited laboratory-induced DNA contamination. Of these five skeletal elements, seven anatomical sampling locations found on four skeletal elements have been determined to be viable alternatives to the destructive sampling of the petrous pyramid11,12. These include the cementum, dentin, and pulp chamber of permanent molars; cortical bone gathered from the superior vertebral notch as well as from the body of thoracic vertebrae; cortical bone stemming from the inferior surface of the apical tuft and shaft of the distal phalanges; and the dense cortical bone along the exterior portion of the tali. While there are several widely applied methods for the sampling of the pars petrosa4,12,13,14, dentin, and the dental pulp chamber1,2,15, published methods describing the successful generation of bone powder from the cementum16, vertebral body, inferior vertebral notch, and talus can be difficult to obtain. As such, here we demonstrate optimized sampling protocols for the petrous pyramid (step 3.1); cementum (step 3.2.1), dentin (step 3.2.2), and dental pulp (step 3.2.3) of adult molars; cortical bone of the vertebral body (step 3.3.1) and superior vertebral arch (step 3.3.2); the distal phalanx (step 3.4); and the talus (step 3.5) in order to make the effective use of these skeletal elements for both aDNA and forensic research more widely accessible.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#16 Dental Drill Bit</td>
			<td>NTI</td>
			<td>H1-016-HP</td>
			<td>example drilling bit</td>
		</tr>
		<tr>
			<td>0.6 mm scroll saw blade</td>
			<td>Fisher Scientific</td>
			<td>50-949-097</td>
			<td>blade for Jewellers Saw</td>
		</tr>
		<tr>
			<td>22mm diamond cutting wheel</td>
			<td>Kahla</td>
			<td>SKU 806 104 358 514 220</td>
			<td>Dremel cutting attachment</td>
		</tr>
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			<td>Commercial Bleach</td>
			<td>Fisher Scientific</td>
			<td>NC1818018</td>
			<td></td>
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			<td>Control Company Ultra-Clean Supreme Aluminum Foil</td>
			<td>Fisher Scientific</td>
			<td>15-078-29X</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA LoBind Tubes (2 mL)</td>
			<td>Eppendorf</td>
			<td>22431048</td>
			<td></td>
		</tr>
		<tr>
			<td>Dremel 225-01 Flex Shaft Attachment</td>
			<td>Dremel</td>
			<td>225-01</td>
			<td>Dremel flexible extension</td>
		</tr>
		<tr>
			<td>Dremel 4300 Rotary Tool</td>
			<td>Dremel</td>
			<td>4300</td>
			<td>Example drill</td>
		</tr>
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			<td>Dremel collet and nut kit</td>
			<td>Dremel</td>
			<td>4485</td>
			<td>Adapters for various Dremel tool attachments/bits</td>
		</tr>
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			<td>Eagle 33 Gallon Red Biohazard Waste Bag</td>
			<td>Fisher Scientific</td>
			<td>17-988-501</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf DNA LoBind 2 mL microcentrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>13-698-792</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (Molecular Biology Grade)</td>
			<td>Millipore Sigma</td>
			<td>1.08543</td>
			<td></td>
		</tr>
		<tr>
			<td>FDA approved level 2 Surgical Mask</td>
			<td>Fisher Scientific</td>
			<td>50-206-0397</td>
			<td>PPE</td>
		</tr>
		<tr>
			<td>Fisherbrand Comfort Nitrile Gloves</td>
			<td>Fisher Scientific</td>
			<td>19-041-171X</td>
			<td>PPE</td>
		</tr>
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			<td>Fisherbrand Safety Glasses</td>
			<td>Fisher Scientific</td>
			<td>19-130-208X</td>
			<td>PPE</td>
		</tr>
		<tr>
			<td>Granger Stationary Vise</td>
			<td>Fisher Scientific</td>
			<td>NC1336173</td>
			<td>benchtop vise</td>
		</tr>
		<tr>
			<td>Invitrogen UltraPure DNase/Rnase free distilled water</td>
			<td>Fisher Scientific</td>
			<td>10-977-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewellers Saw</td>
			<td>Fisher Scientific</td>
			<td>50-949-231</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Sigma-Aldritch</td>
			<td>Z188956</td>
			<td></td>
		</tr>
		<tr>
			<td>Labconco Purifier Logic Biosafety cabinet</td>
			<td>Fisher Scientific</td>
			<td>30-368-1101</td>
			<td></td>
		</tr>
		<tr>
			<td>LookOut DNA Erase</td>
			<td>Millipore Sigma</td>
			<td>L9042-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium weighing boat</td>
			<td>Heathrow Scientific</td>
			<td>HS120223</td>
			<td></td>
		</tr>
		<tr>
			<td>MSC 10pc plier/clamp set</td>
			<td>Fisher Scientific</td>
			<td>50-129-5352</td>
			<td>Miscellaneous&#160;clamps/vise grips for securely holding samples while drilling/cutting</td>
		</tr>
		<tr>
			<td>Sartorius Quintix Semi-Micro Balance</td>
			<td>Fisher Scientific</td>
			<td>14-560-019</td>
			<td>enclosed balance</td>
		</tr>
		<tr>
			<td>Tyvek coveralls with hood</td>
			<td>Fisher Scientific</td>
			<td>01-361-7X</td>
			<td>PPE</td>
		</tr>
		<tr>
			<td>Weigh paper</td>
			<td>Heathrow Scientific</td>
			<td>HS120116</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimized bone sampling protocols for the retrieval of ancient dna from archaeological remains

The methods presented here seek to maximize the chances for the recovery of human DNA from ancient archaeological remains while limiting input sample material. This was done by targeting anatomical sampling locations previously determined to yield the highest amounts of ancient DNA (aDNA) in a comparative analysis of DNA recovery across the skeleton. Prior research has suggested that these protocols maximize the chances for the successful recovery of ancient human and pathogen DNA from archaeological remains. DNA yields were previously assessed by Parker et al. 2020 in a broad survey of aDNA preservation across multiple skeletal elements from 11 individuals recovered from the medieval (radiocarbon dated to a period of circa (ca.) 1040-1400 CE, calibrated 2-sigma range) graveyard at Krakauer Berg, an abandoned medieval settlement near Pei&#223;en Germany. These eight sampling spots, which span five skeletal elements (pars petrosa, permanent molars, thoracic vertebra, distal phalanx, and talus) successfully yielded high-quality ancient human DNA, where yields were significantly greater than the overall average across all elements and individuals. Yields were adequate for use in most common downstream population genetic analyses. Our results support the preferential use of these anatomical sampling locations for most studies involving the analyses of ancient human DNA from archaeological remains. Implementation of these methods will help to minimize the destruction of precious archaeological specimens.

In a separate study11, DNA was extracted from bone powder generated from each anatomical sampling location in 11 individuals, using a standard DNA extraction protocol optimized for short fragments from calcified tissue2. Single-stranded libraries were then produced28 and sequenced on a HiSeq 4000 (75 bp paired-end) to a depth of ~20,000,000 reads per sample. The resulting sequence data was then evaluated for endogenous human DNA content using the EAGER pipeline29 (BWA settings: Seed length of 32, 0.1 mismatch penalty, mapping quality filter of 37). All representative results are reported using the same metrics as Parker et al. 202011 for consistency. Libraries from the powdered portions of the pars petrosa yielded, on average, higher endogenous DNA than any of the other 23 anatomical sampling locations surveyed (Figure 6A-B). The seven additional anatomical sampling locations presented in this protocol (the cementum, first pass of the dental pulp chamber, and dentin of permanent molars; cortical bone from the vertebral body and superior vertebral arch of the thoracic vertebra; cortical bone from the apical tuft of the distal phalanx; and cortical bone from the neck of the talus) produced the next highest yields (with no statistical significance between these anatomical sampling locations; Figure 6A-B; Supplemental File 1: EndogenousDNAPreCap). These alternative locations all consistently produced DNA yields adequate for standard population genetics analyses such as mitochondrial analyses and single nucleotide polymorphism (SNP) analyses. Duplication rates in libraries stemming from all anatomical sampling locations were low (cluster factors &#60; 1.2 on average, calculated as the ratio of all mapping reads to unique mapping reads, Table 2; Supplemental File 1: ClusterFactor), indicating that all libraries screened were of very high complexity. Similarly, average exogenous human DNA contamination estimates were low, averaging &#60; 2% (X chromosome contamination in males, n = 7, as reported by the ANGSD30 pipeline) in all anatomical sampling locations except for the superior vertebral arch (average estimated contamination: 2.11%, with one sample removed as an outlier; KRA005: 19.52%, see Table 2; Supplemental File 1: Xcontamination). Average fragment length (after filtering to remove all reads &#60; 30 bp) was lowest in the material collected from the dental pulp chamber and dentin, with no significant variation among other anatomical sampling locations (55.14 bp and 60.22 bp, respectively in comparison to an average median of 62.87, pair-wise p-values &#60; 0.019, Table 2; Supplemental File 1: AvgFragLength). Additionally, the teeth and thoracic vertebrae each contain multiple anatomical sampling locations where high endogenous DNA recovery was observed, making them particularly suitable as alternatives to the pars petrosa.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63250/63250fig06.jpg" /> 
Figure 6: Human DNA content for all screened samples. Black lines represent the overall mean, while red lines represent the median (solid: human DNA proportion, dashed: mapped human reads per million reads generated). Individual anatomical sampling locations with an average human DNA proportion higher than the overall mean (8.16%) are colorized in all analyses. (A) The proportion of reads mapping to the hg19 reference genome. The blue dashed line represents the theoretical maximum given the pipeline&#39;s mapping parameters (generated using Gargammel31 to simulate a random distribution of 5,000,000 reads from the hg19 reference genome with simulated damage). Individual means (black X) and medians (red circle) are reported for those samples with a higher average human DNA proportion than the overall mean. Confidence intervals indicate upper and lower bounds excluding statistical outliers. (B) The number of unique reads mapping to the hg19 reference genome per million reads of sequencing effort (75 bp paired end). Confidence intervals indicate upper and lower bounds excluding statistical outliers. This figure has been adapted from Parker, C. et al. 202011. <a href="https://www.jove.com/files/ftp_upload/63250/63250fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 2: Average duplication levels (mapping reads/unique reads), average and median fragment lengths, and X chromosome contamination estimates for all anatomical sampling locations. Error reported as the standard error of the mean. This table has been adapted from Parker, C. et al. 202011.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sampling location</td>
			<td>Average duplication factor (# mapped reads /# unique mapped reads)</td>
			<td>Average fragment length in bp</td>
			<td>Average estimated proportion of X chromosome contamination</td>
		</tr>
		<tr>
			<td>Petrous pyramid</td>
			<td>1.188 &#177; 0.006</td>
			<td>65.40 &#177; 1.36</td>
			<td>0.000 &#177; 0.003</td>
		</tr>
		<tr>
			<td>Cementum</td>
			<td>1.197 &#177; 0.028</td>
			<td>67.28 &#177; 1.76</td>
			<td>0.011 &#177; 0.003</td>
		</tr>
		<tr>
			<td>Dentin</td>
			<td>1.188 &#177; 0.061</td>
			<td>60.22 &#177; 2.37</td>
			<td>0.002 &#177; 0.007</td>
		</tr>
		<tr>
			<td>Pulp</td>
			<td>1.179 &#177; 0.024</td>
			<td>55.14 &#177; 2.90</td>
			<td>0.013 &#177; 0.006</td>
		</tr>
		<tr>
			<td>Distal phalanx</td>
			<td>1.191 &#177; 0.049</td>
			<td>65.95 &#177; 1.08</td>
			<td>0.013 &#177; 0.005</td>
		</tr>
		<tr>
			<td>Vertebral body</td>
			<td>1.194 &#177; 0.037</td>
			<td>66.14 &#177; 1.03</td>
			<td>0.008 &#177; 0.003</td>
		</tr>
		<tr>
			<td>Superior vertebral arch</td>
			<td>1.19 &#177; 0.017</td>
			<td>63.02 &#177; 1.23</td>
			<td>0.021 &#177; 0.009*</td>
		</tr>
		<tr>
			<td>Talus</td>
			<td>1.198 &#177; 0.010</td>
			<td>68.20 &#177; 1.24</td>
			<td>0.011 &#177; 0.003</td>
		</tr>
		<tr>
			<td>*Sample KRA005 removed as an outlier at 0.1952</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Code availability 
All analyses programs and R modules used in the analyses of this manuscript are freely available from their respective authors. All custom R code is available by request.
Data availability 
All raw data used in the calculation of representative results is freely available in the European Nucleotide Archive ENA data repository (accession number PRJ-EB36983) or supplemental materials of Parker, C. et al.11.
Supplemental File 1. <a href="https://www.jove.com/files/ftp_upload/63250/SupplementalFile1.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Optimized Bone Sampling Protocols for the Retrieval of Ancient DNA from Archaeological Remains at JoVE.com

Optimized Bone Sampling Protocols for the Retrieval of Ancient DNA from Archaeological Remains

The protocol presents a series of best practice protocols for the collection of bone powder from eight recommended anatomical sampling locations (specific locations on a given skeletal element) across five different skeletal elements from medieval individuals (radiocarbon dated to a period of ca. 1040-1400 CE, calibrated 2-sigma range).

The methods presented here seek to maximize the chances for the recovery of human DNA from ancient archaeological remains while limiting input sample ...

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Genetics

3.5K Views.  Max Planck Institute for the Science of Human History. The protocol presents a series of best practice protocols for the collection of bone powder from eight recommended anatomical sampling locations (specific locations on a given skeletal element) across five different skeletal elements from medieval individuals (radiocarbon dated to a period of ca. 1040-1400 CE, calibrated 2-sigma range).

Video: Optimized Bone Sampling Protocols for the Retrieval of Ancient DNA from Archaeological Remains

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 &#181;g DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., &#62; 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.

Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing.

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics ...

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Protocols for C-Brick DNA Standard Assembly Using Cpf1

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this characteristic, Cpf1 has been used for the establishment of a DNA assembly standard called C-Brick, which has the advantage of long recognition sites and short scars. On a standard C-Brick vector, there are four Cpf1 recognition sites &#8211; the prefix (T1 and T2 sites) and the suffix (T3 and T4 sites) &#8211; flanking biological DNA parts. The cleavage of T2 and T3 sites produces complementary sticky ends, which allow for the assembly of DNA parts with T2 and T3 sites. Meanwhile, a short &#34;GGATCC&#34; scar is generated between parts after assembly. As the newly formed plasmid once again contains the four Cpf1 cleavage sites, the method allows for the iterative assembly of DNA parts, which is similar to those of BioBrick and BglBrick standards. A procedure outlining the use of the C-Brick standard to assemble DNA parts is described here. The C-Brick standard can be widely used by scientists, graduate and undergraduate students, and even amateurs.

CRISPR-associated protein Cpf1 can be guided by a specially designed CRISPR RNA (crRNA) to cleave double-stranded DNA at desired sites, generating sticky ends. Based on this characteristic, a DNA assembly standard (C-Brick) was established, and a protocol detailing its use is described here.

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

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Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

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Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

Single-Molecule F&ouml;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule F&#246;rster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

Single-Molecule F&#246;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Presented here is a protocol for performing single-molecule F&#246;rster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the ...

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a reliable and robust method for genotyping individuals by Variable Number of Tandem Repeat (VNTR) analysis in the context of undergraduate laboratory classes. The human D1S80 VNTR locus is used in this protocol as a highly polymorphic marker based on variation in the number of repetitive sequences.
This simple protocol conveys useful information for teachers and the implementation of DNA fingerprinting in practical laboratory classes. In the presented laboratory exercise, DNA extraction followed by PCR amplification is used to determine genetic variation at the D1S80 VNTR locus. Differences in the fragment size of PCR products are visualized by agarose gel electrophoresis. The fragment sizes and repeat numbers are calculated based on a linear regression of the size and migration distance of a DNA size standard.
Following this guide, students should be able to:
&#8226;&#160; Harvest and extract DNA from buccal mucosa epithelial cells 
&#8226;&#160; Perform a PCR experiment and understand the function of various reaction components 
&#8226;&#160; Analyze the amplicons by agarose gel electrophoresis and interpret the results 
&#8226;&#160; Understand the use of VNTRs in DNA fingerprinting and its application in biological sciences

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

Here we describe a simple protocol to generate DNA fingerprinting profiles by amplifying the VNTR locus D1S80 from epithelial cell DNA.

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a ...