The authors would like to thank Shiping Bo and Shujie Ma for their assistance in NGS data analysis. Funding: this work was supported by the National Key Research and Development Program (Grant No. 2018YFC1003100).

Ethical permission was acquired from the Ethics Committee of Peking University Third Hospital.
1. Preparation
NOTE: The required materials and equipment are listed in Table of Materials.
<ol>
	<li>Reagents
	<ol>
		<li>Prewarm and equilibrate (balanced) 20-30 &#181;L of gamete medium/fertilization medium and cleavage/blastocyst-stage culture medium (covered with mineral oil) and hyaluronidase (in a tightly capped tube) at 37 &#176;C, 5% CO2 and 5% O2 in a Tri-gas incubator overnight before use.</li>
		<li>Prewarm hyaluronidase to 37 &#176;C on a working surface in a fume hood.</li>
		<li>Prepare vitrification buffer and sample collection reagents according to the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Tools
	<ol>
		<li>Prepare sample collection and transfer pipettes (inner diameter of ~200 to 250 &#181;m), denudation/stripper pipettes (inner diameter of &#8805;150 &#181;m, ~130-140 &#181;m, and ~120 &#181;m), and pipettes for washing (inner diameter of ~150 &#181;m) by pulling glass Pasteur pipettes to generate fire-polished open fine tips. 
		&#8203;NOTE: The pipettes used for sample collection/transfer, denudation, and washing can be purchased directly. The holding needles and injection needles can also be purchased directly.</li>
	</ol>
	</li>
</ol>
2. Protocol 1: Sample collection
<ol>
	<li>Pretreatment of oocyte-corona-cumulus complex (OCCCs) before digestion with hyaluronidase
	<ol>
		<li>Achieve ovarian stimulation with both follicle stimulating hormone (FSH) and human menopausal gonadotropin (hMG) preparations. When the lead follicle is &#62;18 mm, use 10,000 IU of chorionic gonadotropin (hCG) for final oocyte maturation.</li>
		<li>Perform oocyte retrieval 36 h after trigger shot. Pick up and transfer oocytes into tissue culture dishes with 2.5 mL of prewarmed m-HTF covered by mineral oil.</li>
		<li>Rapidly transfer the OCCCs to the central well of an organ culture dish containing 1 mL of Fertilization Medium using a transfer pipette and then incubate with the oocytes at 37 &#176;C in a 5% CO2 and 5% O2 incubator for 2-4 h.</li>
	</ol>
	</li>
	<li>Digest OCCCs with hyaluronidase by adding 1 mL of 37 &#176;C prewarmed hyaluronidase (80 IU/mL) to the central well of an organ culture dish containing OCCCs (step 2.1.3). Keep the final concentration of hyaluronidase at 40 IU/mL and mix thoroughly.
	<ol>
		<li>Incubate the OCCCs on a 37 &#176;C thermal platform for 2 min. Observe the changes under a microscope every 30 s until only 1-2 layers of granulosa cells remained.</li>
	</ol>
	</li>
	<li>Denudation of granulosa cells
	<ol>
		<li>Rapidly transfer the digested OCCCs in the culture dish for oocyte handling and cover with mineral oil in each well.</li>
		<li>Observe the separated granulosa cells under a microscope. Gently aspirate and release the oocytes 5 times to remove residual granulosa cells around the oocytes.</li>
		<li>Repeat the previous step in the remaining 3 wells to completely remove the granulosa cells. 
		NOTE: The above steps (2.1-2.3) can be performed according to the routine operation of each laboratory.</li>
	</ol>
	</li>
	<li>Evaluation of the oocyte
	<ol>
		<li>Evaluate the completeness of granulosa cell removal using a microscope. If the cells could not be completely removed, then the retention of 5 or fewer granulosa cells is acceptable at this time. 
		NOTE: If cumulus cells are still attached to the oocyte, the remnant can be removed later on day 3 before the embryo is transferred from cleavage-stage culture medium to blastocyst-stage culture medium.</li>
	</ol>
	</li>
	<li>After performing intracytoplasmic sperm injection (ICSI)26, transfer the oocytes into 20-30 &#181;L cleavage embryo culture medium microdroplets (one oocyte corresponds to one microdroplet) using transfer pipettes and incubate in a 37 &#176;C, 5% CO2 and 5% O2 incubator.
	<ol>
		<li>Record the day of ICSI as day 0. Check the embryos and score according to the Istanbul consensus workshop on embryo assessment of day 1 for fertilization (approximately 18 h), day 2 (approximately 45 h) and day 3 (approximately 68 h) for embryo cleavage27.</li>
	</ol>
	</li>
	<li>Embryo washing
	<ol>
		<li>Prepare 20-30 &#181;L of blastocyst culture medium microdroplets for each embryo covered with mineral oil in tissue culture dishes at day 2 in a 37 &#176;C, 5% CO2 and 5% O2 incubator.</li>
		<li>Prepare another three microdroplets covered with mineral oil, and label the new tissue culture dishes for washing No. 1-3.</li>
		<li>Transfer the day 3 embryos into the washing microdroplets. Gently aspirate and release the embryos 3 times in each droplet using denudation pipettes. 
		NOTE: This procedure can also help remove the residual granular cells attached to the embryo.</li>
		<li>Observe and evaluate the embryos under a microscope on day 3 before the medium was changed from cleavage-stage culture medium to blastocyst culture medium for morphological scoring. If cumulus cells were still attached to the embryo, appropriately pipette up and down in another prewarmed and equilibrated blastocyst culture medium droplet covered with mineral oil with a stripper pipette until the cumulus cells were completely removed. 
		NOTE: All attached cumulus cells had to be removed completely on day 3 before the embryo was transferred from the cleavage-stage culture medium plate to the blastocyst-stage culture medium plate. Any remaining cumulus cells will interfere in the final analysis and give false negative results.</li>
	</ol>
	</li>
	<li>Two options for culture medium collection 
	NOTE: The IVF center can choose from one of two methods for culture medium collection based on the center&#39;s resources, demands, and preferences.
	<ol>
		<li>Option 1: Embryo wash and culture 
		NOTE: This option is for IVF labs that perform vitrification on the morning of day 5.
		<ol>
			<li>Transfer the embryo into prewarmed (37 &#176;C) microdroplets of culture medium, and gently wash each embryo serially in 3 microdroplets by pipetting at day 4 afternoon.</li>
			<li>Transfer each embryo into a unique prewarmed (37 &#176;C) single microdroplet of culture medium for sample collection. The volume of a single droplet of culture medium cannot exceed 25 &#181;L.</li>
			<li>Perform blastocyst embryo culture on day 5/day 6 at 37 &#176;C, 5% CO2, and 5% O2.</li>
		</ol>
		</li>
		<li>Option 2: Embryo wash and culture 
		&#8203;NOTE: This option is for IVF labs that perform vitrification on day 5 afternoon or day 6.
		<ol>
			<li>Transfer the embryo into prewarmed (37 &#176;C) microdroplets of 10-15 &#181;L culture medium, and gently wash each embryo serially in 3 microdroplets by pipetting on day 5.</li>
			<li>Transfer each embryo into a unique prewarmed (37 &#176;C) single microdroplet of culture medium for sample collection. The volume of a single droplet of culture medium cannot exceed 15 &#181;L.</li>
			<li>Perform blastocyst embryo culture on day 5/day 6 at 37 &#176;C and 5% CO2.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Sample Collection
	<ol>
		<li>Gently adjust the ICM at a considerable distance from the targeted point of the laser beam, which focuses on the cell junction of the trophectoderm to generate a small hole in the trophectoderm to release the fluid from the blastocoel cavity.&#160;Then embryos are moved to freezing solution for cryopreservation&#160; according to the conventional process.</li>
		<li>Transfer culture medium from each cultured embryo into an RNase/DNase-free PCR tube containing 5 &#181;L of cell lysis buffer.</li>
		<li>Collect the same amount of culture medium without being used for embryo culture as a negative control. Freeze all collected samples immediately in liquid nitrogen and then store at &#8722;80 &#176;C after being collected until subjected to the NICS assay.</li>
	</ol>
	</li>
	<li>Perform vitrification as described in the protocol.</li>
</ol>
3. Protocol 2: Library Construction
<ol>
	<li>Culture medium lysis
	<ol>
		<li>Dilute 1 &#181;L of positive control (10 ng human gDNA) with 199 &#181;L of fresh culture medium. Mix thoroughly and centrifuge the tube briefly (200 x g for 5 s).</li>
		<li>Transfer 10 &#181;L of day 5-day 6 blastocyst culture medium, diluted positive control, and fresh culture medium to new 0.2 mL PCR tubes.</li>
		<li>Add 1 &#181;L of MT Enzyme Mix to each PCR tube and mix thoroughly by pipetting and centrifuge immediately for 2-3 s at 200 x g.</li>
		<li>Put the PCR tube(s) from step 3.1.3 in a preheated NICS Sample Prep Station and run the lysis program as follows: 10 min at 75 &#176;C; 4 min at 95 &#176;C; hold at 22 &#176;C. 
		NOTE: The sample prep station is comparable to a standard PCR machine.
		<ol>
			<li>Click the Lysis icon to enter the setup screen.</li>
			<li>Select Tube for Control mode; input 10 &#181;L for Sample volume; select On for Hotlid control and enter 105 &#176;C for the temperature. Select No for Pause at the first seg. Click OK to proceed.</li>
			<li>Wait until Remain time shows --:--:--, which indicates the end of the program, and then click Stop to terminate the program.</li>
		</ol>
		</li>
		<li>Stop the program after the process is complete. Proceed to the next step immediately.</li>
	</ol>
	</li>
	<li>Pre-Library Preparation
	<ol>
		<li>Thaw the Pre-Lib Buffer to RT. Mix thoroughly by pipetting and centrifuge immediately for 2-3 s at 200 x g.</li>
		<li>Prepare a master mix for prelibrary reaction as follows: add 2 &#181;L of Pre-Lib Enzyme Mix to 60 &#181;L of Pre-Lib Buffer, mix the reaction thoroughly and centrifuge briefly.</li>
		<li>Add 60 &#181;L of prelibrary reaction mix into each pretreated medium sample from the previous step. Mix thoroughly by pipetting and centrifuge immediately for 2-3 s at 200 x g.</li>
		<li>Place the PCR tube(s) from step 3.2.3 in the Sample Prep Station and run the prelibrary program as follows: 95 &#176;C for 2 min; 12 cycles of 15 &#176;C for 40 s, 22 &#176;C for 40 s, 33 &#176;C for 30 s, 65 &#176;C for 30 s, 72 &#176;C for 40 s, 95 &#176;C for 10 s and 63 &#176;C for 10 s; and hold at 4 &#176;C.
		<ol>
			<li>Click the Pre_Lib icon to enter the setup screen.</li>
			<li>Select Tube for Control mode; input 70 &#181;L for Sample volume; select On for Hot lid control and enter 105 &#176;C for the temperature. Select No for Pause at the first seg. Click OK to proceed.</li>
			<li>Wait until Remain time shows --:--:--, which indicates the end of the program, and click Stop to terminate the program.</li>
		</ol>
		</li>
		<li>Stop the program when the process is complete. Proceed to the next step immediately.</li>
	</ol>
	</li>
	<li>Library Preparation
	<ol>
		<li>Thaw the Library Buffer to RT. Mix thoroughly by pipetting and centrifuge immediately for 2-3 s at 200 x g.</li>
		<li>Prepare a master mix for library reaction as follows: add 1.6 &#181;L of Library Enzyme Mix to 60 &#181;L of Library Buffer, mix the reaction thoroughly and centrifuge briefly.</li>
		<li>Add 60 &#181;L of library reaction mix and 2 &#181;L of Barcode Primer to each prelibrary product from step 3.2.3. Mix the reaction thoroughly and centrifuge briefly.</li>
		<li>Place the PCR tube(s) from step 3.2.3 in the thermal cycler and run library preparation program as follows: 94 &#176;C for 30 s; 17 cycles of 94 &#176;C for 25 s, 62 &#176;C for 30 s, and 72 &#176;C for 45 s); and then hold at 4 &#176;C.
		<ol>
			<li>Click the Lib_Prep icon to enter the setup screen.</li>
			<li>Select Tube for Control mode; input 130 &#181;L for Sample volume; select On for Hotlid control and enter 105 &#176;C for the corresponding temperature. Select No for Pause at the first seg. Click OK to proceed.</li>
			<li>Wait until Remain time shows --:--:--, which indicates the end of the program, and click Stop to terminate the program.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Library Purification
	<ol>
		<li>Remove Magbeads from storage at 2-8 &#176;C for at least 20 min before the purification step. Vortex and mix the Magbeads for 20 s. Dispense enough beads for the purification step into a new 1.5 mL microcentrifuge tube and warm beads to RT.</li>
		<li>Add 1x Magbeads into each library. Mix by pipetting up and down &#8805;10 times and incubate at RT for 5 min. 
		NOTE: For example, add 100 &#181;L of Magbeads to 100 &#181;L of library sample.</li>
		<li>After incubation, centrifuge the tube briefly and place on a magnetic stand.</li>
		<li>Wait for approximately 5 min until the solution becomes clear. While keeping the tube on the magnetic stand, carefully aspirate the solution and discard.</li>
		<li>Add 200 &#181;L of freshly prepared 80% ethanol to the tube. Incubate at RT for 30 s and carefully remove the supernatant. Repeat one more time.</li>
		<li>Remove the ethanol as completely as possible. Air dry the beads on the magnetic stand for approximately 5-10 min at RT.</li>
		<li>Remove the tube from the magnetic stand, add 17.5 &#181;L of elution buffer, and vortex the tube to resuspend the beads. Centrifuge the tube briefly and incubate at RT for 5 min.</li>
		<li>Place the tube onto the magnetic stand and wait until the solution becomes clear. Carefully transfer 15 &#181;L supernatant to a new tube.</li>
	</ol>
	</li>
	<li>Library quantification
	<ol>
		<li>Quantify purified libraries by using the fluorometer according to the user guide of the qubit dsDNA HS Assay kits28. The yield of the libraries ranges from ~15 to 300 ng.</li>
	</ol>
	</li>
	<li>Library Pooling
	<ol>
		<li>Use 10 nanograms of each library sample for pooling.</li>
	</ol>
	</li>
	<li>Sequencing
	<ol>
		<li>Refer to the sequencing user guide (15027617 v01)29.</li>
		<li>Purified library sequences of 50 bp at a single end on the platform yielded approximately 2 million reads for each sample, and a 0.03 &#215; sequencing depth was recommended.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Enter the users&#39; Name and Password in the login page</li>
		<li>After logging into the system, click Analysis and a new page will appear. Click Create Submission under the NICS-A tab. Then, choose NGS for the platform, select corporation, choose NICSInst&#160;for the reagent, enter the project information in the box under Project ID, set the analysis preferences and upload the files. Once all sequencing files are successfully uploaded, click Submit to start the analysis (Figure 3A).</li>
		<li>Click View Submissions to show the list of submitted projects. Once the analysis is complete, the status of a project will become Completed and a Show button will appear in the report field. Click the Show button to view the summary table of NICS analysis (Figure 3B).</li>
		<li>Click the Export report button to save the reports (Figure 3C). 
		NOTE: Three types of files will be exported for each analysis. A graphic file that includes all copy number variation (CNV) plots for each chromosome and whole genome, which will be stored under the &#34;graph&#34; folder; a spreadsheet that contains the sample QC details of this analysis run; a document file that contains the NICS reports customized by the user; and a spreadsheet that contains the sample summary information of this analysis run.</li>
	</ol>
	</li>
</ol>

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L., Scaruffi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+DNA+content+in+embryo+culture+medium+is+significantly+associated+with+human+embryo+fragmentation.">Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation.</a> Human Reproduction. 28 (10), 2652-2660 (2013).</li><li>Stigliani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+DNA+in+Day+3+embryo+culture+medium+is+a+novel,+non-invasive+biomarker+of+blastocyst+potential+and+implantation+outcome.">Mitochondrial DNA in Day 3 embryo culture medium is a novel, non-invasive biomarker of blastocyst potential and implantation outcome.</a> Molecular Human Reproduction. 20 (12), 1238-1246 (2014).</li><li>Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medium-Based+Noninvasive+Preimplantation+Genetic+Diagnosis+for+Human+&#945;-Thalassemias-SEA.">Medium-Based Noninvasive Preimplantation Genetic Diagnosis for Human &#945;-Thalassemias-SEA.</a> Medicine. 94 (12), e669 (2015).</li><li>Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+chromosome+screening+of+human+embryos+by+genome+sequencing+of+embryo+culture+medium+for+in+vitro+fertilization.">Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization.</a> Proceedings of the National Academy of Sciences. 113 (42), 11907-11912 (2016).</li><li>Capalbo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+efficacy+of+blastocoel+fluid+and+spent+media+as+sources+of+DNA+for+preimplantation+genetic+testing+in+standard+clinical+conditions.">Diagnostic efficacy of blastocoel fluid and spent media as sources of DNA for preimplantation genetic testing in standard clinical conditions.</a> Fertility and Sterility. 110 (5), 870-879 (2018).</li><li>Tobler, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blastocoel+fluid+from+differentiated+blastocysts+harbors+embryonic+genomic+material+capable+of+a+whole-genome+deoxyribonucleic+acid+amplification+and+comprehensive+chromosome+microarray+analysis.">Blastocoel fluid from differentiated blastocysts harbors embryonic genomic material capable of a whole-genome deoxyribonucleic acid amplification and comprehensive chromosome microarray analysis.</a> Fertility and Sterility. 104 (2), 418-425 (2015).</li><li>Magli, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preimplantation+genetic+testing:+polar+bodies,+blastomeres,+trophectoderm+cells,+or+blastocoelic+fluid?.">Preimplantation genetic testing: polar bodies, blastomeres, trophectoderm cells, or blastocoelic fluid?.</a> Fertility and Sterility. 105 (3), 676-683 (2016).</li><li>Kuznyetsov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+novel+non-invasive+preimplantation+genetic+screening+approach.">Evaluation of a novel non-invasive preimplantation genetic screening approach.</a> PLoS One. 13 (5), e0197262 (2018).</li><li>Li, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preimplantation+Genetic+Screening+with+Spent+Culture+Medium/Blastocoel+Fluid+for+in+Vitro+Fertilization.">Preimplantation Genetic Screening with Spent Culture Medium/Blastocoel Fluid for in Vitro Fertilization.</a> Scientific Reports. 8 (1), 9275 (2018).</li><li>Jiao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+invasive+preimplantation+genetic+testing+using+blastocyst+culture+medium.">Minimally invasive preimplantation genetic testing using blastocyst culture medium.</a> Human Reproduction. 34 (7), 1369-1379 (2019).</li><li>Palermo, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Births+after+intracytoplasmic+injection+of+sperm+obtained+by+testicular+extraction+from+men+with+nonmosaic+Klinefelter's+syndrome.">Births after intracytoplasmic injection of sperm obtained by testicular extraction from men with nonmosaic Klinefelter's syndrome.</a> New England Journal of Medicine. 338 (9), 588-590 (1998).</li><li>Alpha Scientists in Reproductive, M., &#38; Embryology, E. S. I. G. o. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Istanbul+consensus+workshop+on+embryo+assessment:+proceedings+of+an+expert+meeting.">The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting.</a> Human Reproduction. 26 (6), 1270-1283 (2011).</li><li>. Qubit dsDNA HS Assay Kit Available from: <a target="_blank" href="https://www.thermofisher.com/order/catalog/product/Q32851?ICID=search-product">https://www.thermofisher.com/order/catalog/product/Q32851?ICID=search-product</a> (2015)</li><li>. Miseq system use guide Available from: <a target="_blank" href="https://support.illumina.com/downloads/miseq_system">https://support.illumina.com/downloads/miseq_system</a> (2016)</li><li>Lane, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ability+to+detect+aneuploidy+from+cell+free+DNA+collected+from+media+is+dependent+on+the+stage+of+development+of+the+embryo.">Ability to detect aneuploidy from cell free DNA collected from media is dependent on the stage of development of the embryo.</a> Fertility and Sterility. 108 (3), (2017).</li><li>Rubio, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+prospective+study+of+concordance+between+embryonic+cell-free+DNA+and+trophectoderm+biopsies+from+1301+human+blastocysts.">Multicenter prospective study of concordance between embryonic cell-free DNA and trophectoderm biopsies from 1301 human blastocysts.</a> American Journal of Obstetrics and Gynecology. 223 (5), 751-751 (2020).</li><li>Rubio, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+cell-free+DNA+versus+trophectoderm+biopsy+for+aneuploidy+testing:+concordance+rate+and+clinical+implications.">Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: concordance rate and clinical implications.</a> Fertility and Sterility. 112 (3), 510-519 (2019).</li><li>Lledo, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consistent+results+of+non-invasive+PGT-A+of+human+embryos+using+two+different+techniques+for+chromosomal+analysis.">Consistent results of non-invasive PGT-A of human embryos using two different techniques for chromosomal analysis.</a> Reproductive BioMedicine Online. 42 (3), 555-563 (2021).</li><li>Kuznyetsov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+Invasive+Cell-Free+Human+Embryo+Aneuploidy+Testing+(miPGT-A)+Utilizing+Combined+Spent+Embryo+Culture+Medium+and+Blastocoel+Fluid+-Towards+Development+of+a+Clinical+Assay.">Minimally Invasive Cell-Free Human Embryo Aneuploidy Testing (miPGT-A) Utilizing Combined Spent Embryo Culture Medium and Blastocoel Fluid -Towards Development of a Clinical Assay.</a> Scientific Reports. 10 (1), 7244 (2020).</li></ol>

Yaxin Yao, Jieliang Ma, Jing Wang and Sijia Lu are employees of Yikon Genomics Co., Ltd.

Modifications and troubleshooting
If the NICS results are contaminated with parental genetic materials, make sure all cumulus-corona radiata cells are removed and make sure ICSI is performed for fertilization. Improper medium storage or template preparation processes are avoided, which may degrade DNA. The working space was purified thoroughly with DNase and RNase decontamination reagents. To avoid contamination from other embryos, one embryo was always cultured in a single dr...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62619">Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis</a>. The Protocol and Representaive Results sections were&#160;updated.
In the Protocol, step 3.8.2 was updated from:
After logging into the system, click Create Submission under the NICS tab. Then, select the sequencing platform, choose ChromInst for the reagent, enter the project information in the box under Project ID, set the analysis preferences and upload the files. Once all sequencing files are successfully uploaded, click Submit to start the analysis (Figure 3A).
to:
After logging into the system, click Create Submission under the NICS-A tab. Then, choose NGS for the platform, select corporation, choose ChromInst for the reagent, enter the project information in the box under Project ID, set the analysis preferences and upload the files. Once all sequencing files are successfully uploaded, click Submit to start the analysis (Figure 3A).
In the Representative Results, Figure 3 was updated from:
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62619/62619fig03.jpg" /> 
Figure 3. Data Analysis. (A) The page of Create Submission. There are different options for the user application. For sequencing platform, users can choose Illumina or Ion Torrent. For analysis criterion, there are two length detection resolution for selection, the whole chromosome and whole arm level. The users also can choose whether the mosaicism or gender information is reported. Finished the above parameter setting,click on the box under File upload and choose the appropriate sequencing files to upload. For Illumina, choose the files with an extension of fastq.gz. For Ion Torrent platform, choose files with an extension of bam. Click Submit to start the analysis after successfully upload. (B) The view of summary table. The summary table consists of following information: Sample Name: The name of each NICS sample is listed; Data QC: Indicates whether the sequencing file passes the QC for NICS analysis; Conclusion: Indicates whether the NICS analysis is normal or abnormal, &#34;N/A&#34; indicates no conclusive result is available; Gender: If the user chooses to report the sex information, this column will appear in the summary table; Karyotype: Shows the analysis results; CNV plot (Whole Genome): View the CNV profiles of all chromosomes; CNV plot (By Chromosome): View the CNV profiles of each chromosome. (C) The Save Report Page. Click Export report button next to the Summary of Results. Select the information you want to show on the final report and click Export. Select Save File in the appearing dialog window and then click OK. The reports will be saved to the Download folder of the computer. <a href="https://www.jove.com/files/ftp_upload/62619/62619fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62619/62619fig3v2.jpg" /> 
Figure 3. Data Analysis. (A) There are different options for the user application. For sequencing platform corporation, users can choose Illumina, Ion Torrent or MGI. The users can choose whether the gender information is reported. Finished the above parameter setting, click on the box under File upload and choose the appropriate sequencing files to upload. For Illumina, choose the files with an extension of fastq.gz. Click Submit&#160;to start the analysis after successfully upload. (B) The view of summary table. The summary table consists of following information: Sample Name: The name of each NICS sample is listed; Data QC: Indicates whether the sequencing file passes the QC for NICS analysis; AI Rating: The rating (A, B or C) for each NICS sample; AI_Rating Interpretation: Evaluation of embryo implantation potential; AI Grading: The score for each NICS sample; CNV plot (Whole Genome): View the CNV profiles of all chromosomes; (C) The Save Report Page. Click Export report&#160;button next to the Summary of Results. Select the information you want to show on the final report and click Export. The reports will be saved to the Download&#160;folder of your computer. <a href="https://www.jove.com/files/ftp_upload/62619/62619fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62619">Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis</a>. The Protocol and Representaive Results sections were&#160;updated.

Erratum: Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis

Assisted reproductive technologies (ARTs) have been increasingly used for the treatment of infertility. However, the success rate of ART, such as IVF, has been limited, and the pregnancy loss rate is significantly higher than that of the normal population1. The main cause of these problems is chromosomal abnormalities, which commonly exist in preimplantation human embryos2. PGT-A is an effective method of screening embryos for chromosomal balance before implantation3,4. Some studies have proven that PGT-A can reduce the rate of abortion and improve the rate of pregnancy5,6,7,8. However, PGT-A requires complex technical expertise that requires specific training and experience. The invasive embryo biopsy procedure could also potentially cause damage to the embryos9. Studies have shown that blastomere biopsy can hinder subsequent development, and the number of biopsied TEs may affect the implantation rates10. Although the long-term biosafety issue of embryo biopsy has not yet been evaluated thoroughly in humans, animal studies have shown its negative influences on embryo development11,12,13.
Previous reports indicated that trace amounts of DNA materials were secreted into the culture medium during embryo development, and efforts have been made to perform comprehensive chromosome screening (CCS) using spent embryo culture medium14,15,16,17,18. However, the detection rates and the accuracy of the tests have not met the requirements for extensive clinical use. The present study reported an improvement in the NICS assay for increasing the detection rates as well as the accuracy of the NICS test19. In recent years, blastocoele fluid (BF) has been studied as an analytical sample of minimally invasive PGT-A. However, the proportion of successful genome-wide amplification and detectable DNA in blastocyst fluid samples ranges from 34.8% to 82%20,21,22. The volume of BF reported in various studies ranges from 0.3 nL to 1 &#181;L. In view of the low amount of DNA in BF, it is possible to increase the amount of cell-free DNA by mixing blastocyst fluid and culture medium to improve the success rate and consistency of detection. Kuznyetsov et al.23 and Li et al.24 treated the zona pellucida with a laser and released blastocyst fluid into the culture medium to improve the total amount of embryonic DNA, and the amplification rate of the combined medium/BF samples after WGA was 100% and 97.5%, respectively. Jiao et al.25 also obtained a 100% amplification success rate by using the same method.
The present study reports a detailed protocol that includes spent media sample preparation, NGS preparation and data analysis. By carefully removing cumulus cells from oocytes, the present study performed intracytoplasmic single sperm injection (ICSI) and blastocyst culture. The, day 4-day 5/day 6 spent medium was collected for WGA and NGS library preparation. By using NICS technology, the present study streamlined the WGA and NGS library preparation steps in approximately 3 h and obtained CCS results noninvasively in approximately 9 h.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL EP tube, 0.2 mL PCR tube</td>
			<td>Axygen</td>
			<td>MCT-150-C, PCR-02-C</td>
			<td>DNase/RNase free, Low Binding PCR tubes and 1.5 mL micro-centrifuge tubes are recommended.</td>
		</tr>
		<tr>
			<td>10 &#181;L, 200 &#181;L, 1000 &#181;L DNase /RNase Free Tips</td>
			<td>Axygen</td>
			<td>T-300-R-S, T-200-Y-R-S, T-1000-B-R-S</td>
			<td>This can be replaced by other brand/For sample transfer</td>
		</tr>
		<tr>
			<td>100 % ethanol</td>
			<td>Sinopharm Chemical</td>
			<td>10009218</td>
			<td>This can be replaced by other brand/For DNA library purification</td>
		</tr>
		<tr>
			<td>Barcode Primer1-48</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>For library amplificaton</td>
		</tr>
		<tr>
			<td>BD Falcon Organ Culture Dish, Sterile</td>
			<td>BD Bioscience</td>
			<td>363037</td>
			<td>This can be replaced by other brand/For embryo culture</td>
		</tr>
		<tr>
			<td>BD Falcon Tissue culture Dishes (Easy Grip) , Sterile</td>
			<td>BD Bioscience</td>
			<td>353001</td>
			<td>This can be replaced by other brand/For embryo culture</td>
		</tr>
		<tr>
			<td>BD Falcon Tissue culture Dishes, Sterile</td>
			<td>BD Bioscience</td>
			<td>353002</td>
			<td>This can be replaced by other brand/For embryo culture</td>
		</tr>
		<tr>
			<td>Cell Lysis Buffer</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>For culture medium pre-treatment</td>
		</tr>
		<tr>
			<td>Cell Lysis Enzyme</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>For culture medium pre-treatment</td>
		</tr>
		<tr>
			<td>ChromGo software</td>
			<td>Yikon Genomics</td>
			<td></td>
			<td>Data analysis</td>
		</tr>
		<tr>
			<td>CMPure Magbeads</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>For library purification</td>
		</tr>
		<tr>
			<td>Cryotop open systerm</td>
			<td>&#160;KITAZATO BioPharma</td>
			<td>81110</td>
			<td>This can be replaced by other brand/For embryo vitrification</td>
		</tr>
		<tr>
			<td>Distill water</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>To dissolve DNA</td>
		</tr>
		<tr>
			<td>ES (Vitrification kit)</td>
			<td>&#160;KITAZATO BioPharma</td>
			<td>Reagent inVitrification kit</td>
			<td>This can be replaced by other brand/For embryo vitrification</td>
		</tr>
		<tr>
			<td>HOLDNIG</td>
			<td>ORIGIO</td>
			<td>MPH-MED-35</td>
			<td>This can be replaced by other 
			brand/For ICSI</td>
		</tr>
		<tr>
			<td>Hyaluronidase solution, 80 U/mL</td>
			<td>SAGE</td>
			<td>ART4007-A</td>
			<td>This can be replaced by other brand/Digest oocyte-corona-cumulus complex</td>
		</tr>
		<tr>
			<td>ICSI</td>
			<td>ORIGIO</td>
			<td>MPH-35-35</td>
			<td>This can be replaced by other brand/For ICSI</td>
		</tr>
		<tr>
			<td>Illumina MiSeq&#174; System</td>
			<td>Illumina</td>
			<td>SY-410-1001</td>
			<td>For library sequencing</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Labotect</td>
			<td>Inkubator C16</td>
			<td>This can be replaced by other brand/For embryo culture</td>
		</tr>
		<tr>
			<td>Library buffer</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>For library amplificaton</td>
		</tr>
		<tr>
			<td>Library Enzyme Mix</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>For library amplificaton</td>
		</tr>
		<tr>
			<td>Magnetic Stand</td>
			<td>DynaMagTM-2</td>
			<td>12321D</td>
			<td>For library purification</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>OLYMPUS</td>
			<td>1X71</td>
			<td>This can be replaced by other brand/For embryo observation</td>
		</tr>
		<tr>
			<td>Mini-centrifuge</td>
			<td>ESSENSCIEN</td>
			<td>ELF6</td>
			<td>For separation</td>
		</tr>
		<tr>
			<td>MT Enzyme Mix</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>For culture medium pre-treatment</td>
		</tr>
		<tr>
			<td>NICSInst library preparation kit</td>
			<td>Yikon Genomics</td>
			<td>KT1000800324</td>
			<td>Whole genome amplification and library construction</td>
		</tr>
		<tr>
			<td>NICSInst Sample Prep Station</td>
			<td>Yikon Genomics</td>
			<td>&#160;ME1001003</td>
			<td>Amplificate DNA</td>
		</tr>
		<tr>
			<td>Nunc IVF 4-Well Dish</td>
			<td>Thermo Scientific</td>
			<td>144444</td>
			<td>This can be replaced by other brand/For embryo washing and blastocyst culture</td>
		</tr>
		<tr>
			<td>Pasteur Pipette</td>
			<td>Oirgio</td>
			<td>&#160;MXL3-IND-135</td>
			<td>This can be replaced by other brand/For embryo tansfer</td>
		</tr>
		<tr>
			<td>Pasteur pipettes</td>
			<td>ORIGIO</td>
			<td>PP-9-1000</td>
			<td>This can be replaced by other brand/For IVF laboratory</td>
		</tr>
		<tr>
			<td>Pre-Lib Buffer</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>Pre-library preparation</td>
		</tr>
		<tr>
			<td>Pre-Lib Enzyme</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>Pre-library preparation</td>
		</tr>
		<tr>
			<td>Qubit&#174; 3.0 Fluorometer</td>
			<td>Thermo Scientific</td>
			<td>Q33216</td>
			<td>For library quantification</td>
		</tr>
		<tr>
			<td>Quinn&#39;s Advantage Blastocyst Medium</td>
			<td>SAGE</td>
			<td>ART-1029</td>
			<td>For embryo blastocyst stage culture</td>
		</tr>
		<tr>
			<td>Quinn&#39;s Advantage Cleavage Medium</td>
			<td>SAGE</td>
			<td>ART-1026</td>
			<td>This can be replaced by other brand/For embryo cleavage stage culture</td>
		</tr>
		<tr>
			<td>Quinn&#39;s Advantage Fertilization Medium</td>
			<td>SAGE</td>
			<td>ART-1020</td>
			<td>This can be replaced by other brand/For oocyte and sperm fertilization</td>
		</tr>
		<tr>
			<td>Quinn&#39;s Advantage m-HTF Medium with HEPES</td>
			<td>SAGE</td>
			<td>ART-1023</td>
			<td>This can be replaced by other brand/For embryo clutrure</td>
		</tr>
		<tr>
			<td>Quinn&#39;s Advantage SPS Serum protein Substitute Kit</td>
			<td>SAGE</td>
			<td>ART-3010</td>
			<td>This can be replaced by other brand/To denude the oocyte</td>
		</tr>
		<tr>
			<td>Quinn&#39;s Advantage Tissue culture mineral oil</td>
			<td>SAGE</td>
			<td>ART-4008P</td>
			<td>This can be replaced by other brand/To cover the culture medium</td>
		</tr>
		<tr>
			<td>STRIPPER TIPS</td>
			<td>ORIGIO</td>
			<td>MXL3-IND-135</td>
			<td>This can be replaced by other brand/For denudating granulosa cells</td>
		</tr>
		<tr>
			<td>Vitrification Cryotop Open systerm</td>
			<td>KIZTAZATO</td>
			<td>81111</td>
			<td>This can be replaced by other brand/For embryo vitrification</td>
		</tr>
		<tr>
			<td>Vitrification kit</td>
			<td>&#160;KITAZATO BioPharma</td>
			<td>VT101</td>
			<td>This can be replaced by other brand/For embryo vitrification</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Qilinbeier</td>
			<td>DNYS8</td>
			<td>Sample mix</td>
		</tr>
		<tr>
			<td>VS (Vitrification kit)</td>
			<td>&#160;KITAZATO BioPharma</td>
			<td>Reagent inVitrification kit</td>
			<td>This can be replaced by other brand/For embryo vitrification</td>
		</tr>
		<tr>
			<td>ZILOS-tk Laser System</td>
			<td>Hamilton Thorne</td>
			<td>CLASS 1 laser</td>
			<td>This can be replaced by other brand/For artificial blastocoele collapse</td>
		</tr>
	</tbody>
</table>

chromosome screening of human preimplantation embryos by using spent culture medium: sample collection and chromosomal ploidy analysis

In clinical in vitro fertilization (IVF), the prevailing method for PGT-A requires biopsy of a few cells from the trophectoderm (TE). This is the lineage that forms the placenta. This method, however, requires specialized skills, is invasive, and suffers from false positives and negatives because the chromosome numbers in the TE and the inner cell mass (ICM), which develops into the fetus, are not always the same. NICS, a technology requiring sequencing of DNA that released into the culture medium from both TE and ICM, may offer a way out to these problems but has previously been shown to have limited efficacy. The present study reports the full protocol of NICS, which includes culture medium sampling methods, whole genome amplification (WGA) and library preparation, and NGS data analysis by analysis software. Considering the different cryopreservation times in different embryo laboratories, embryologists have two methods for collecting embryo culture medium that can be selected according to the actual conditions of the IVF laboratory.

The present study applied the proposed method to a patient. IRB approval and informed consent were obtained before the application of NICS analysis. The present study obtained 6 blastocysts from patients and performed NICS on all 6 embryos on day 4 to day 5 medium. Chromosome abnormalities caused by the parents&#39; balanced translocation were detected in five of chromosomes with the NICS assay; therefore, they could not be used for transfer (Figure 4A-E). The NICS results o...

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Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis

The present study reports a protocol for chromosome screening of human embryos that uses spent culture medium, which avoids embryo biopsy and enables chromosome ploidy identification using NGS. The present article presents the detailed procedure, including the preparation of culture medium, whole genome amplification (WGA), next-generation sequencing (NGS) library preparation, and data analysis.

In clinical in vitro fertilization (IVF), the prevailing method for PGT-A requires biopsy of a few cells from the trophectoderm (TE). This is the lineage ...

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2.0K Views.  Peking University Third Hospital. The present study reports a protocol for chromosome screening of human embryos that uses spent culture medium, which avoids embryo biopsy and enables chromosome ploidy identification using NGS. The present article presents the detailed procedure, including the preparation of culture medium, whole genome amplification (WGA), next-generation sequencing (NGS) library preparation, and data analysis.

Video: Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Expedited Radiation Biodosimetry by Automated Dicentric Chromosome Identification (ADCI) and Dose Estimation

Biological radiation dose can be estimated from dicentric chromosome frequencies in metaphase cells. Performing these cytogenetic dicentric chromosome assays is traditionally a manual, labor-intensive process not well suited to handle the volume of samples which may require examination in the wake of a mass casualty event. Automated Dicentric Chromosome Identifier and Dose Estimator (ADCI) software automates this process by examining sets of metaphase images using machine learning-based image processing techniques. The software selects appropriate images for analysis by removing unsuitable images, classifies each object as either a centromere-containing chromosome or non-chromosome, further distinguishes chromosomes as monocentric chromosomes (MCs) or dicentric chromosomes (DCs), determines DC frequency within a sample, and estimates biological radiation dose by comparing sample DC frequency with calibration curves computed using calibration samples. This protocol describes the usage of ADCI software. Typically, both calibration (known dose) and test (unknown dose) sets of metaphase images are imported to perform accurate dose estimation. Optimal images for analysis can be found automatically using preset image filters or can also be filtered through manual inspection. The software processes images within each sample and DC frequencies are computed at different levels of stringency for calling DCs, using a machine learning approach. Linear-quadratic calibration curves are generated based on DC frequencies in calibration samples exposed to known physical doses. Doses of test samples exposed to uncertain radiation levels are estimated from their DC frequencies using these calibration curves. Reports can be generated upon request and provide summary of results of one or more samples, of one or more calibration curves, or of dose estimation.

Expedited Radiation Biodosimetry by Automated Dicentric Chromosome Identification ADCI and Dose Estimation

The cytogenetic dicentric chromosome (DC) assay quantifies exposure to ionizing radiation. The Automated Dicentric Chromosome Identifier and Dose Estimator software accurately and rapidly estimates biological dose from DCs in metaphase cells. It distinguishes monocentric chromosomes and other objects from DCs, and estimates biological radiation dose from the frequency of DCs.

Biological radiation dose can be estimated from dicentric chromosome frequencies in metaphase cells. Performing these cytogenetic dicentric chromosome ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60&#8722;80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2&#8722;4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented ...

Microsatellite DNA Genotyping and Flow Cytometry Ploidy Analyses of Formalin-fixed Paraffin-embedded Hydatidiform Molar Tissues

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by excessive trophoblastic proliferation and abnormal embryonic development. There are two types of HM based on microscopic morphological evaluation, complete HM (CHM) and partial HM (PHM). These can be further subdivided based on the parental contribution to the molar genomes. Such characterization of HM, by morphology and genotype analyses, is crucial for patient management and for the fundamental understanding of this intriguing pathology. It is well documented that morphological analysis of HM is subject to wide interobserver variability and is not sufficient on its own to accurately classify HM into CHM and PHM and distinguish them from hydropic non-molar abortions. Genotyping analysis is mostly performed on DNA and tissues from formalin-fixed paraffin-embedded (FFPE) products of conception, which have less than optimal quality and may consequently lead to wrong conclusions. In this article, detailed protocols for multiplex genotyping and flow cytometry analyses of FFPE molar tissues are provided, along with the interpretation of the results of these methods, their troubleshooting, and integration with the morphological evaluation, p57KIP2 immunohistochemistry, and fluorescence in situ hybridization (FISH) to reach a correct and robust diagnosis. Here, the authors share the methods and lessons learned in the past 10 years from the analysis of approximately 400 products of conception.

Hydatidiform moles are abnormal human pregnancies with heterogeneous aetiologies that can be classified according to their morphological features and parental contribution to the molar genomes. Here, protocols of multiplex microsatellite DNA genotyping and flow cytometry of formalin-fixed paraffin-embedded molar tissues are described in detail, together with results&#8217; interpretation and integration.

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by excessive trophoblastic proliferation and abnormal embryonic development. There are ...

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported for in vitro differentiation of keratinocytes cultured in two-dimensional (2D) submerged manners using various induction conditions. Described here is a procedure for 2D in vitro keratinocyte differentiation method by contact inhibition and subsequent molecular characterization by RNA-seq. In brief, keratinocytes are grown in defined keratinocyte medium supplemented with growth factors until they are fully confluent. Differentiation is induced by close contacts between the keratinocytes and further stimulated by excluding growth factors in the medium. Using RNA-seq analyses, it is shown that both 1) differentiated keratinocytes exhibit distinct molecular signatures during differentiation and 2) the dynamic gene expression pattern largely resembles cells during epidermal stratification. As for comparison to normal keratinocyte differentiation, keratinocytes carrying mutations of the transcription factor p63 exhibit altered morphology and molecular signatures, consistent with their differentiation defects. In conclusion, this protocol details the steps for 2D in vitro keratinocyte differentiation and its molecular characterization, with an emphasis on bioinformatic analysis of RNA-seq data. Because RNA extraction and RNA-seq procedures have been well-documented, it is not the focus of this protocol. The experimental procedure of in vitro keratinocyte differentiation and bioinformatic analysis pipeline can be used to study molecular events during epidermal differentiation in healthy and diseased keratinocytes.

Presented here is a stepwise procedure for in vitro differentiation of human primary keratinocytes by contact inhibition followed by characterization at the molecular level by RNA-seq analysis.

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported ...