This work was supported by the National Science and Technology Council of Taiwan. [NSTC 111-2314-B-006-003]

1. Poly(A) RNA purification
NOTE: Use the total RNA treated with DNase I and examine the total RNA quality and integrity by capillary or conventional gel electrophoresis assessment before proceeding to poly(A) RNA purification. Investigators should be able to identify the 28S and 18S rRNA ribosomal bands in the high molecular weight field and the 5.8S rRNA band in the low molecular weight field without any significant smear bands in the electropherogram. The purification steps essentially follow the manufacturer&#39;s instructions with minor modifications indicated in the specific steps. See the Table of Materials for details related to all materials and instruments used in this protocol.
<ol>
	<li>Preparation
	<ol>
		<li>Warm up the reagent, washing buffer, oligo(dT) beads, and lysis buffer at room temperature for 30 min before conducting the following procedures.</li>
		<li>For the isolation of poly(A)-enriched RNA, prepare 10-20 &#181;g of an aliquot of high-quality total RNA as the starting material by transferring an adequate quantity of RNA into a nuclease-free 1.5 mL microcentrifuge tube. Incubate in the heat block at 70 &#176;C for 5 min, then keep on ice for 2 min. 
		NOTE: The quantities of total RNA as the starting material should be appropriately adjusted depending on the desired quantity of mRNA and the nature of the cells used. Empirically, the poly(A)-enriched RNA may account for 1-5% abundances in the total RNA. An aliquot of 10 &#181;g of high-quality total RNA, should provide high-quality poly(A)-enriched RNA in the 100-500 ng range for the downstream bisulfite conversion.</li>
		<li>Meanwhile, fully resuspend the oligo(dT) beads. Transfer the required amounts of beads suspension to a new 1.5 mL microcentrifuge tube, then place on the magnet for 3 min till the magnetic beads form a pellet. 
		NOTE: Use 50 &#181;L oligo(dT) beads for 10 &#181;g of total RNA input. The volume of beads in this step could be further optimized depending on the cells used.</li>
		<li>Remove the supernatant and resuspend the oligo(dT) beads with equal amounts (the volume of the beads suspension used in step 1.1.3.) of lysis buffer and place on the magnet for 3 min before removing the supernatant. 
		NOTE: Do not overdry the beads; the user should quickly proceed to step 1.2.1.</li>
	</ol>
	</li>
	<li>The first round of purification
	<ol>
		<li>Add lysis buffer to the RNA sample tube (volume ratio 4:1) and mix thoroughly before transferring to the oligo(dT) beads. 
		&#8203;NOTE: If the RNA sample volume is 20 &#181;L, then add 80 &#181;L of lysis buffer.</li>
		<li>Resuspend the RNA: lysis buffer mix and oligo(dT) beads fully by pipetting at least 10x.</li>
		<li>Allow the samples to incubate with continuous rotation for 15 min at room temperature.</li>
		<li>Place the sample on the magnet for 5 min or until the supernatant is clear; discard the supernatant.</li>
		<li>Add 600 &#181;L of Wash Buffer 1 to the beads and carefully mix to resuspend the beads.</li>
		<li>Place the tube on the magnet for 5 min or until the supernatant is clear, discard the supernatant, and repeat wash step 1.2.5 once.</li>
		<li>Wash the beads by adding 300 &#181;L of Wash Buffer 2, and gently resuspend the beads.</li>
		<li>Place the tube on the magnet for 5 min or until the supernatant is clear, discard the supernatant, and repeat wash step 1.2.7 once.</li>
		<li>Add 30 &#181;L of cold 10 mM Tris-HCl (Elution buffer) to the sample tube and incubate at 70 &#176;C for 5 min.</li>
		<li>Quickly transfer the tube onto the magnet, and when the suspension is clear after 20 s or more, transfer the supernatant containing eluted poly(A) enriched RNA to the new 1.5 mL microcentrifuge tube.</li>
	</ol>
	</li>
	<li>The second round of purification
	<ol>
		<li>Add 120 &#181;L (4x the volume of eluted sample) lysis buffer to the eluted RNA sample and mix thoroughly.</li>
		<li>Wash the beads used in the first round of purification with an equal amount of lysis buffer as step 1.1.4, pipet 10x to resuspend the beads, and then place them on the magnet for 5 min before discarding the supernatant.</li>
		<li>Transfer the RNA: lysis buffer mix to the washed beads and mix thoroughly by pipetting at least 10x.</li>
		<li>Incubate the sample with continuous rotation for 15 min at room temperature.</li>
		<li>Repeat steps 1.2.4 to 1.2.8 to remove salt and other RNA subtypes. 
		NOTE: The volume of washing buffer can be reduced to 300 &#181;L of Wash Buffer 1 and 150 &#181;L of Wash Buffer 2.</li>
		<li>Add 25 &#181;L (desired volume) of cold 10 mM Tris-HCl (Elution buffer) to the sample tube and incubate at 70 &#176;C for 5 min.</li>
		<li>Quickly transfer the tube onto the magnet, and when the suspension is clear after 20 s or more, transfer the supernatant containing eluted poly(A) enriched RNA to a new 1.5 mL microcentrifuge tube.</li>
		<li>Transfer 2.2 &#181;L to 8-strip tubes for quality assessment of RNA quantity and quality using RNA high-sensitivity assays.</li>
		<li>Store the sample at -20 &#176;C for short periods and -70 &#176;C for a longer period. 
		NOTE: This can be a pause point in the protocol.</li>
	</ol>
	</li>
</ol>
2. Bisulfite conversion
NOTE: The centrifugation steps were all performed at room temperature. The procedures are essentially performed accordingly to the manufacturer&#39;s instructions but with an additional step 2.2 for adding the spike-in control mRNA sequence(s) before the bisulfite reaction step 2.3.
<ol>
	<li>Transfer 19 &#181;L of purified poly(A) enriched RNA sample to a 0.2 mL 8-strip tube.</li>
	<li>Add 1.0 &#181;L of spike-in control, which is free from any modifications at the predetermined 1:10,000 quantity ratio (i.e., 0.1 ng of luciferase mRNA: 1 &#181;g of purified poly(A) enriched RNA) into the RNA sample of step 2.1. 
	NOTE: The spike-in control sequence can be Firefly luciferase RNA, Renilla luciferase, or in vitro transcribed RNA sequence with no methylated cytosines.</li>
	<li>Add 130 &#181;L of conversion regent to the tube and gently invert several times for thorough mixing.</li>
	<li>Place the tube in the PCR machine and perform three cycles of heating at 70 &#176;C for 10 min followed by incubating at 64 &#176;C for 45 min each and then a final step to cool down to 4 &#176;C.</li>
	<li>Place the column onto the collection tube and transfer 250 &#181;L of RNA binding buffer to the column.</li>
	<li>Transfer the sample from step 2.4&#160;to the column from step 2.5 and pipet thoroughly.</li>
	<li>Add 400 &#181;L of 95-100% of ethanol to the column, quickly close the cap, and invert several times.</li>
	<li>Centrifuge at 10,000 &#215; g for 30 s at 25 &#176;C and discard the flowthrough.</li>
	<li>Add 200 &#181;L of RNA wash buffer to the column.</li>
	<li>Centrifuge at 10,000 &#215; g for 30 s at 25 &#176;C and discard the flowthrough.</li>
	<li>Add 200 &#181;L of desulfonation buffer and incubate at room temperature for 30 min.</li>
	<li>Centrifuge at 10,000 &#215; g for 30 s at 25 &#176;C and discard the flowthrough.</li>
	<li>Add 400 &#181;L of RNA wash buffer and centrifuge at 10,000 &#215; g for 30 s at 25 &#176;C and discard the flowthrough. Repeat this step once.</li>
	<li>Centrifuge again at 10,000 &#215; g for 2 min at 25 &#176;C to completely remove the residual wash buffer. 
	NOTE: The other centrifugation step 2.14 for 2 min before elution is to completely remove the wash buffer. The wash step is performed twice to wash out the high-salt component from the bisulfite reagent and the desulfonation buffer on the column.</li>
	<li>Transfer the column to a new 1.5 mL microcentrifuge tube.</li>
	<li>Add 20-30 &#181;L of Nuclease-free H2O to the column and stand at room temperature for 1 min.</li>
	<li>Centrifuge at 10,000 &#215; g for 30 s at 25 &#176;C.</li>
	<li>Transfer 2.2 &#181;L to 8-strip tubes for the assessment of RNA quantity and quality.</li>
	<li>Store the bisulfite-treated RNA sample at -20 &#176;C for short periods and -70 &#176;C for a longer period. 
	&#8203;NOTE: This can be a pause point in the protocol.</li>
</ol>
3. Bisulfite-treated mRNA library preparation
NOTE: Follow the library preparation instruction protocol section 4 for use with purified mRNA or rRNA-depleted RNA. The first priming step should follow the FFPE RNA protocol since the bisulfite treatment fragmentizes the RNA. Perform every step in the laminar flow hood and add the reaction mixture on an ice-chilled cooling rack..
<ol>
	<li>Priming the input RNA
	<ol>
		<li>Transfer the desired amount of bisulfite-treated mRNA sample in a total maximum of 5 &#181;L or add Nuclease-free H2O to make a total of 5 &#181;L. 
		NOTE: A minimum of 10 ng of bisulfite-treated mRNA as input is recommended for this type of RNA library preparation and to generate the final molarity of the 4nM library product.</li>
		<li>Add 1 &#181;L of Random primer (lilac) to the sample and mix by pipetting.</li>
		<li>Place the sample tube in the PCR machine preset at 65 &#176;C and lid at 105 &#176;C for 5 min and hold at 4 &#176;C.</li>
	</ol>
	</li>
	<li>First-strand cDNA synthesis
	<ol>
		<li>Add 4 &#181;L of synthesis reaction buffer (lilac), 8 &#181;L of strand specificity reagent (brown), and 2 &#181;L of synthesis enzyme mix (brown) to the sample, which makes a total volume of 20 &#181;L. Mix thoroughly by pipetting at least 10x and quickly spin down.</li>
		<li>Place the sample tube in the PCR machine preset at 25 &#176;C for 10 min, 42 &#176;C for 50 min, 70 &#176;C for 15 min, and hold at 4 &#176;C. 
		NOTE: For inserts over 200 bases, a 42 &#176;C incubation period of 50 min is suggested; for insert size below 200 bases, a 42 &#176;C incubation period of 15 min is suggested.</li>
	</ol>
	</li>
	<li>Second-strand cDNA synthesis
	<ol>
		<li>Add 8 &#181;L of synthesis reaction buffer with dUTP mix (orange), 4 &#181;L of synthesis enzyme mix (orange), and 48 &#181;L of Nuclease-free H2O. Mix thoroughly by pipetting at least 10x and quickly spin down.</li>
		<li>Place the sample tube in the PCR machine preset at 16 &#176;C for 1 h with the lid set off or &#8804;40 &#176;C.</li>
	</ol>
	</li>
	<li>Purification of double-stranded cDNA with DNA purification beads
	<ol>
		<li>Prewarm the DNA purification beads 30 min before conducting the purification and vortex to resuspend the beads.</li>
		<li>Add 144 &#181;L of resuspended DNA purification beads to the cDNA sample from step 3.3.2&#160;and mix thoroughly by pipetting at least 10x.</li>
		<li>Incubate at room temperature for 5 min.</li>
		<li>Place the sample tube on a magnetic rack for 5 min until the solution is clear and then, remove the supernatant and keep the beads that contain DNA.</li>
		<li>Add 200 &#181;L of freshly made 80% ethanol to the tube with the beads while keeping the sample on the magnetic rack and incubate at room temperature for 30 s.</li>
		<li>Remove the supernatant and repeat step 3.4.5&#160;once, but this time carefully remove all the residual supernatant.</li>
		<li>Air dry the beads for 1-5 min while keeping the sample on the magnet. 
		NOTE: Do not overdry the beads; the color should be dark brown.</li>
		<li>Remove the sample from the magnetic rack and elute the DNA by adding 53 &#181;L of 0.1x TE (10 mM Tris-HCl and 1 mM EDTA, pH 8.0) buffer to the beads. Mix thoroughly by pipetting at least 10x and incubate at room temperature for 2 min.</li>
		<li>Place the tube onto the magnetic rack for 5 min or until the solution is clear.</li>
		<li>Transfer 50 &#181;L of supernatant containing double-stranded cDNA to a new 8-strip tube. 
		NOTE: The protocol can be stopped at this point and the sample can be stored at -30 &#176;C.</li>
	</ol>
	</li>
	<li>End preparation of cDNA library
	<ol>
		<li>Add 7 &#181;L of end-polishing Reaction Buffer (green) and 3 &#181;L of the end-polishing Enzyme Mix (green) to the eluted double-stranded cDNA sample.</li>
		<li>Mix the mixture by pipetting 10x with a 50 &#181;L volume setting without creating bubbles, then quickly spin down the sample.</li>
		<li>Place the sample in a PCR machine and incubate at 20 &#176;C for 30 min, 65 &#176;C for 30 min, and hold at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Adaptor ligation
	<ol>
		<li>Dilute the adaptor (red) according to the manufacturer&#39;s instructions.</li>
		<li>Add 2.5 &#181;L of the diluted adaptor, 1 &#181;L of ligation enhancer (red), and 30 &#181;L of Ligation Master Mix (red) to the sample.</li>
		<li>Mix the mixture by pipetting 10x with an 80 &#181;L volume setting without creating bubbles, then quickly spin down the sample.</li>
		<li>Place the sample in a PCR machine and incubate at 20 &#176;C for 15 min.</li>
		<li>Add 3 &#181;L of the mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII (blue) to the sample and pipet thoroughly.</li>
		<li>Place back the sample in the PCR machine and incubate at 37 &#176;C for 15 min with the lid set to 75 &#176;C.</li>
	</ol>
	</li>
	<li>Purification of ligation reaction treated cDNAs 
	NOTE: The size selection with SPRIselect Beads, or optionally with AMpure XP beads can both be used for this purpose18.
	<ol>
		<li>Prewarm the beads 30 min before conducting the purification and vortex to resuspend them.</li>
		<li>Transfer 25 &#181;L of resuspended beads to the cDNA sample from step 3.6.6 and mix thoroughly by pipetting at least 10x.</li>
		<li>Incubate at room temperature for 5 min.</li>
		<li>Place the sample tube on a magnetic rack for 5 min until the solution is clear and then transfer the supernatant to a new 8-strip tube and discard the beads.</li>
		<li>Add 10 &#181;L of resuspended beads to the supernatant and mix thoroughly by pipetting at least 10x.</li>
		<li>Place the sample tube on a magnetic rack for 5 min until the solution is clear and then discard the supernatant and do not disturb the beads.</li>
		<li>Add 200 &#181;L of freshly made 80% ethanol to the tube with the beads while keeping the sample on the magnetic rack and incubate at room temperature for 30 s.</li>
		<li>Remove the supernatant and repeat step 3.7.7 once, but this time carefully remove all the residual supernatant.</li>
		<li>Air-dry the beads for 1-5 min while keeping the sample on the magnet. 
		&#8203;NOTE: Do not overdry the beads; the color should be dark brown.</li>
		<li>Remove the sample from the magnetic rack and elute the DNA by adding 17 &#181;L of 0.1x TE buffer to the beads. Mix thoroughly by pipetting at least 10x and incubate at room temperature for 2 min.</li>
		<li>Place the tube onto the magnetic rack for 5 min until the solution is clear.</li>
		<li>Transfer 15 &#181;L of supernatant, which contains target end-prepared dsDNA to a new 8-strip tube.</li>
	</ol>
	</li>
	<li>PCR enrichment of adaptor-ligated DNA
	<ol>
		<li>Add 25 &#181;L of master mix (blue), 5 &#181;L of 5&#39; adaptor primer, and 5 &#181;L of the 3&#39; adaptor primer to the adaptor-ligated DNA sample. Mix thoroughly by pipetting at least 10x.</li>
		<li>Place the sample in the PCR machine and perform PCR amplification using the following steps: (1) 98 &#176;C for 30 s, (2) 98 &#176;C for 10 s, (3) 65 &#176;C for 75 s, (4) 65 &#176;C for 5 min, and (5) hold at 4 &#176;C; in which the steps (2) and (3) should repeat for an N+2 cycle number where N equals to the RNA input quantity in the instruction manual. This means 2 additional cycles are needed due to the double-side size selection of samples performed in step 3.7. 
		NOTE: For instance, 8 ng of the purified mRNA input would be recommended with 9-10 cycles of PCR; but with double-side, size-selected, ligated cDNA in step 3.7, the recommended PCR cycle would be 11-12 cycles.</li>
	</ol>
	</li>
	<li>Purification of PCR-amplified libraries
	<ol>
		<li>Prewarm the DNA purification beads for 30 min before conducting the purification and vortex to resuspend the beads.</li>
		<li>Transfer 45 &#181;L (0.9x) of resuspended beads to the PCR product from step 3.8.2 and mix thoroughly by pipetting at least 10x.</li>
		<li>Incubate at room temperature for 5 min.</li>
		<li>Place the sample tube on a magnetic rack for 5 min until the solution is clear and then remove the supernatant and keep the beads that contain DNA.</li>
		<li>Add 200 &#181;L of freshly made 80% ethanol to the tube with the beads, leave the sample on the magnetic rack and allow it to stand at room temperature for 30 s.</li>
		<li>Remove the supernatant and repeat step 3.9.5 once, but this time carefully remove all the residual supernatant.</li>
		<li>Air-dry the beads for 1-5 min while keeping the sample on the magnet. 
		NOTE: Do not overdry the beads; the color should be dark brown.</li>
		<li>Take the sample off the magnetic rack and add 22 &#181;L of 0.1x TE buffer to the beads to elute the DNA. Mix thoroughly by pipetting at least 10x and incubate at room temperature for 2 min.</li>
		<li>Place the tube onto the magnetic rack and allow it to stand for 5 min or until the solution is clear at room temperature.</li>
		<li>Transfer 20 &#181;L of supernatant to a new 8-strip tube.</li>
		<li>Transfer 2.2 &#181;L to 8-strip tubes for quality assessment of library quantity and quality. 
		NOTE: The quantity of the library can also be detected by qPCR (see the Table of Materials).</li>
		<li>Store the bsRNA-seq library sample at -20 &#176;C.</li>
	</ol>
	</li>
</ol>

Creating Robust Bisulfite-mRNA Libraries for Transcriptome-Wide Mapping

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Nucleic Acids. 26, 575-593 (2021).</li><li>Saplaoura, E., Perrera, V., Colot, V., Kragler, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylated+RNA+Immunoprecipitation+Assay+to+Study+m5C+Modification+in+Arabidopsis.">Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis.</a> Journal of Visualized Experiments:JoVE. (159), e61231 (2020).</li><li>Quail, M. A., Swerdlow, H., Turner, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+protocols+for+the+illumina+genome+analyzer+sequencing+system.">Improved protocols for the illumina genome analyzer sequencing system.</a> Current Protocols in Human Genetics. , (2009).</li><li>Chen, S. -. Y., 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=RNA+bisulfite+sequencing+reveals+NSUN2-mediated+suppression+of+epithelial+differentiation+in+pancreatic+cancer.">RNA bisulfite sequencing reveals NSUN2-mediated suppression of epithelial differentiation in pancreatic cancer.</a> Oncogene. 41 (22), 3162-3176 (2022).</li><li>Squires, J. E., 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=Widespread+occurrence+of+5-methylcytosine+in+human+coding+and+non-coding+RNA.">Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA.</a> Nucleic Acids Research. 40 (11), 5023-5033 (2012).</li><li>Han, X., 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=Dynamic+DNA+5-hydroxylmethylcytosine+and+RNA+5-methycytosine+reprogramming+during+early+human+development.">Dynamic DNA 5-hydroxylmethylcytosine and RNA 5-methycytosine reprogramming during early human development.</a> Genomics, Proteomics &amp; Bioinformatics. , (2022).</li><li>Amort, T., 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=Long+non-coding+RNAs+as+targets+for+cytosine+methylation.">Long non-coding RNAs as targets for cytosine methylation.</a> RNA biology. 10 (6), 1003-1008 (2013).</li><li>Sun, Z., 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=Effects+of+NSUN2+deficiency+on+the+mRNA+5-methylcytosine+modification+and+gene+expression+profile+in+HEK293+cells.">Effects of NSUN2 deficiency on the mRNA 5-methylcytosine modification and gene expression profile in HEK293 cells.</a> Epigenomics. 11 (4), 439-453 (2019).</li><li>Huang, T., Chen, W., Liu, J., Gu, N., Zhang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+identification+of+mRNA+5-methylcytosine+in+mammals.">Genome-wide identification of mRNA 5-methylcytosine in mammals.</a> Nature Structural and Molecular Biology. 26 (5), 380-388 (2019).</li><li>Rieder, D., Amort, T., Kugler, E., Lusser, A., Trajanoski, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=meRanTK:+methylated+RNA+analysis+ToolKit.">meRanTK: methylated RNA analysis ToolKit.</a> Bioinformatics. 32 (5), 782-785 (2015).</li><li>Kraus, A. J., Brink, B. G., Siegel, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+specific+oligo-based+depletion+of+rRNA.">Efficient and specific oligo-based depletion of rRNA.</a> Scientific Reports. 9 (1), 12281 (2019).</li><li>Edelheit, S., Schwartz, S., Mumbach, M. R., Wurtzel, O., Sorek, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome-wide+mapping+of+5-methylcytidine+RNA+modifications+in+bacteria,+archaea,+and+yeast+reveals+m(5)C+within+archaeal+mRNAs.">Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m(5)C within archaeal mRNAs.</a> PLoS Genetics. 9 (5), e1003602 (2013).</li><li>Furlan, M., 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=Computational+methods+for+RNA+modification+detection+from+nanopore+direct+RNA+sequencing+data.">Computational methods for RNA modification detection from nanopore direct RNA sequencing data.</a> RNA Biology. 18, 31-40 (2021).</li><li>Leger, A., 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=RNA+modifications+detection+by+comparative+Nanopore+direct+RNA+sequencing.">RNA modifications detection by comparative Nanopore direct RNA sequencing.</a> Nature Communications. 12 (1), 7198 (2021).</li><li>Mateos, P. A., 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=Identification+of+m6A+and+m5C+RNA+modifications+at+single-molecule+resolution+from+Nanopore+sequencing.">Identification of m6A and m5C RNA modifications at single-molecule resolution from Nanopore sequencing.</a> bioRxiv. , (2022).</li><li>Johnson, Z., Xu, X., Pacholec, C., Xie, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+evaluation+of+parameters+in+RNA+bisulfite+sequencing+data+generation+and+analysis.">Systematic evaluation of parameters in RNA bisulfite sequencing data generation and analysis.</a> NAR Genomics and Bioinformatics. 4 (2), 045 (2022).</li><li>Zhang, Z., 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=Systematic+calibration+of+epitranscriptomic+maps+using+a+synthetic+modification-free+RNA+library.">Systematic calibration of epitranscriptomic maps using a synthetic modification-free RNA library.</a> Nature Methods. 18 (10), 1213-1222 (2021).</li><li>Chao, H. -. P., 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=Systematic+evaluation+of+RNA-Seq+preparation+protocol+performance.">Systematic evaluation of RNA-Seq preparation protocol performance.</a> BMC Genomics. 20 (1), 571 (2019).</li></ol>

The authors have no conflicts of interest to disclose.

In this protocol, a detailed pipeline of poly(A) enrichment, bisulfite conversion, and library preparation was achieved by utilizing standardized components. Further sequencing analysis provided the identification of mRNA 5-methylcytosine in samples of interest.
The critical step is the quality of starting material-total RNA-since the degradation of RNA would impact the recovery rate of poly(A) RNA purification. The sample should be carefully handled and RNase contamination avoided before cond...

Among over 150 types of post-transcriptional modifications1, 5-methylcytosine (m5C) modification has been identified in various types of RNAs, including ribosomal RNA, transfer RNA, messenger RNA, micro RNA, long non-coding RNA, vault RNA, enhancer RNA, and small cajal body-specific RNAs2. The RNA m5C is associated with diverse biological and pathological mechanisms such as regulating plant root development3, viral gene expression4, and cancer progression5. The aim of this protocol is to provide streamlined pipelines to characterize the transcriptome-wide mRNA m5C modification profile of biological samples in different developmental stages or in the disease setting. Transcriptome-wide bisulfite-sequencing was developed to characterize the positions and the quantitative cytosine methylation levels in the bisulfite-converted RNA at base-pair resolution6,7,8,9. This is particularly useful when studying the association of m5C with gene expression and RNA fate that is involved in the biological regulatory mechanisms in cells. In the mammalian cell, there are two known m5C readers: ALYREF can recognize m5C at the nucleus and serves as an mRNA nucleus-to-cytosol transporter10, while YBX1 can recognize m5C in the cytoplasm and increase mRNA stabilization11. Aberrant m5C mRNAs related to immune pathways were reported in Systemic Lupus Erythematosus CD4+ T cells12. Studies have revealed an association between mRNA m5C modification and modulation of cancer immunity and cancer progression13,14. Hence, mapping the m5C modification profile on the mRNA can provide crucial information to elucidate the potential regulatory machinery.
To investigate the functional roles of RNA m5C modification under certain biological conditions, the bisulfite conversion-based (bsRNA-seq) and antibody affinity enrichment-based approaches such as m5C-RIP-seq, miCLIP-seq, and 5-Aza-seq can be combined with the high-throughput sequencing platform to provide efficient detection of targeted regions and sequences with the m5C modifications on a transcriptome-wide scale15,16. The advantage of this protocol provides the comprehensive RNA m5C landscape at a single-base resolution since the antibody affinity enrichment-based approach relies upon the availability of high-quality antibodies and could achieve the single-fragment resolution of m5C methylation landscape17.
All RNA samples will be processed with two rounds of mRNA enrichment using oligo(dT) beads, three cycles of bisulfite reaction, and the sequencing library preparation. To monitor the RNA quality, each RNA sample will be examined by capillary gel electrophoresis before and after the procedures of mRNA purification and bisulfite reaction to assess fragment distribution. The purified libraries will be examined by their PCR amplicon qualities, DNA size distribution fragments by capillary gel electrophoresis, and their overall quantities examined by fluorescence dyes-based quantitative assays before sequencing. The system can also be used to analyze a broad spectrum of biological samples such as agricultural produce, isolated virions, cell lines, model organisms, and pathological specimens.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agilent 2100 Electrophoresis Bioanalyzer System</td>
			<td>Agilent, Santa Clara, CA</td>
			<td></td>
			<td>RNA quality detection</td>
		</tr>
		<tr>
			<td>AMpure XP beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>purify DNA</td>
		</tr>
		<tr>
			<td>Bioanalyzer DNA high sensitivity kit</td>
			<td>Agilent, Santa Clara, CA</td>
			<td>5067-4626</td>
			<td>DNA quality dection</td>
		</tr>
		<tr>
			<td>Bioanalyzer RNA 6000 Pico kit</td>
			<td>Agilent, Santa Clara, CA</td>
			<td>5067-1513</td>
			<td>RNA quality dection</td>
		</tr>
		<tr>
			<td>DiaMag02 - magnetic rack</td>
			<td>Diagenode, Denville, NJ</td>
			<td>B04000001</td>
			<td>assist library preparation</td>
		</tr>
		<tr>
			<td>DiaMag1.5 - magnetic rack</td>
			<td>Diagenode, Denville, NJ</td>
			<td>B04000003</td>
			<td>assist poly(A) RNA purificaion</td>
		</tr>
		<tr>
			<td>Dynabeads mRNA DIRECT purification kit</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>61011</td>
			<td>poly(A) RNA purificaion; Wash Buffer 1 and Wash Buffer 2</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>J.T.Baker</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ RNA methylation kit</td>
			<td>Zymo, Irvine, CA</td>
			<td>R5002</td>
			<td>bisulfite treatment</td>
		</tr>
		<tr>
			<td>Firefly luciferase mRNA</td>
			<td>Promega, Madison, WI, USA</td>
			<td>L4561</td>
			<td>spike in control seqeunce&#160;</td>
		</tr>
		<tr>
			<td>KAPA Library Quantification Kits</td>
			<td>Roche, Switzerland</td>
			<td>KK4824</td>
			<td>library quantification</td>
		</tr>
		<tr>
			<td>Nanodrop spectrophotometer</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td></td>
			<td>Total RNA quantity detection</td>
		</tr>
		<tr>
			<td>NEBNext multiplex Oligos for illumina (index Primer set1)</td>
			<td>New England Biolabs, Ipswich, MA</td>
			<td>E7335S</td>
			<td>library preparation</td>
		</tr>
		<tr>
			<td>NEBNext Ultra <img alt="Equation 1" src="/files/ftp_upload/65352/65352eq01.jpg" style="margin: auto;" /> Directional RNA Library Prep Kit for Illumina</td>
			<td>New England Biolabs, Ipswich, MA</td>
			<td>E7760S</td>
			<td>library preparation</td>
		</tr>
		<tr>
			<td>Nuclease-free Water</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>P2 pipetman</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>4641010</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 2.0 fluorometer&#160;</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td></td>
			<td>RNA quantity detection</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>Q32854</td>
			<td>DNA quantity detection</td>
		</tr>
		<tr>
			<td>Qubit RNA HS Assay Kit</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>Q32852</td>
			<td>RNA quantity detection</td>
		</tr>
	</tbody>
</table>

enrichment of mrna and bisulfite-mrna library preparation for next-generation sequencing

RNA post-transcriptional modifications in various types of RNA transcripts are associated with diverse RNA regulation in eukaryotic cells. Aberrant RNA 5-methylcytosine modifications and the dysregulated expression of RNA methyltransferases have been shown to be associated with various diseases, including cancers. Transcriptome-wide bisulfite-sequencing was developed to characterize the positions and the quantitative cytosine methylation levels in the bisulfite-converted RNA at the base-pair resolution. Herein, this protocol presents the procedures of two rounds of poly(A) RNA purification, three cycles of bisulfite reaction, and library preparation in detail to allow the transcriptome-wide mapping of mRNA 5-methylcytosine modification sites. The assessment of RNA quantity and quality after the main reaction is essential to monitor RNA integrity and is a critical step for ensuring high-quality sequencing libraries. Ideally, the procedures can be completed within three days. With this protocol, using high-quality total RNA as the input can practically build up robust bisulfite-mRNA libraries for next-generation sequencing from the sample of interest.

A series of bsRNA-seq libraries from cell lines19 were generated by following the procedures in this report (Figure 1). After total RNA purification accompanied by DNase treatment performed on cell line samples and the quality checked by gel electrophoresis and UV-Vis spectrophotometry (A260/A280), the RNA sample can proceed to poly(A) RNA enrichment. To determine whether the double purification could remove the majority of ribosomal RNA, the purifica...

Watch this Scientific Journal Video about Decoding RNA Methylation's Role in Pancreatic Cancer - A Single-Base Resolution Study at JoVE.com

Author Spotlight: Decoding RNA Methylation's Role in Pancreatic Cancer - A Single-Base Resolution Study 

This protocol provides an easy-to-follow workflow to conduct poly(A) RNA purification, bisulfite conversion, and library preparation using standardized equipment for a biological sample of interest.

Enrichment of mRNA and Bisulfite-mRNA Library Preparation for Next-Generation Sequencing

RNA post-transcriptional modifications in various types of RNA transcripts are associated with diverse RNA regulation in eukaryotic cells. Aberrant RNA ...

This research aims to study the role of RNA methylation, specifically 5-methylcytosine modification, in pancreatic cancer. The questions we want to answer are how upregulation or downregulation of certain RNA methyltransferase affects cancer cell phenotypes and also, through which RNA substrates. This study provides the comprehensive RNA m5C landscape at a single-base resolution with standardized equipment.From this study using bisulfite RNA seq, the results links the association of RNA m5C modification with poor differentiation of cancer cells. This should pave the way for future studies in discerning passenger versus disease driver effects of specific m5C in the RNA transcripts. With this technique, we can identify the set of differentially-methylated RNAs that may participate in tumor progression.Further, single molecule base experiments can provide a more detailed regulatory role of specific RNA m5C.

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965 Views.  National Cheng Kung University. This research aims to study the role of RNA methylation, specifically 5-methylcytosine modification, in pancreatic cancer. The questions we want to answer are how upregulation or downregulation of certain RNA methyltransferase affects cancer cell phenotypes and also, through which RNA substrates. This study provides the comprehensive RNA m5C landscape at a single-base resolution with standardized equipment.From this study using bisulfite RNA seq, the results links the association of RN...

Video: Author Spotlight: Decoding RNA Methylation's Role in Pancreatic Cancer - A Single-Base Resolution Study 

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

3' End Sequencing Library Preparation with A-seq2

Studies in the last decade have revealed a complex and dynamic variety of pre-mRNA cleavage and polyadenylation reactions. mRNAs with long 3&#39; untranslated regions (UTRs) are generated in differentiated cells whereas proliferating cells preferentially express transcripts with short 3&#39;UTRs. We describe the A-seq protocol, now at its second version, which was developed to map polyadenylation sites genome-wide and study the regulation of pre-mRNA 3&#39; end processing. Also this current protocol takes advantage of the polyadenylate (poly(A)) tails that are added during the biogenesis of most mammalian mRNAs to enrich for fully processed mRNAs. A DNA adaptor with deoxyuracil at its fourth position allows the precise processing of mRNA 3&#39; end fragments for sequencing. Not including the cell culture and the overnight ligations, the protocol requires about 8 h hands-on time. Along with it, an easy-to-use software package for the analysis of the derived sequencing data is provided. A-seq2 and the associated analysis software provide an efficient and reliable solution to the mapping of pre-mRNA 3&#39; ends in a wide range of conditions, from 106 or fewer cells.

3&#039; End Sequencing Library Preparation with A-seq2

This protocol describes a method for mapping pre-mRNA 3&#39; end processing sites.

Studies in the last decade have revealed a complex and dynamic variety of pre-mRNA cleavage and polyadenylation reactions. mRNAs with long 3&#39; ...

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 &#946;-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called &#34;domainome&#34;, will be well expressed and soluble, making them suitable for structural/functional studies.
By cloning and displaying the &#34;domainome&#34; directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification.
The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data analysis.

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...

Assessing Genomic Alteration in Non-Squamous Non-Small Cell Lung Cancer Patients

Ultra-Fast Amplicon-Based Next-Generation Sequencing in Non-Squamous Non-Small Cell Lung Cancer

The number of molecular alterations to be tested for targeted therapy of non-squamous non-small cell lung cancer (NS-NSCLC) patients has significantly increased these last few years. The detection of molecular abnormalities is mandatory for the optimal care of advanced or metastatic NS-NSCLC patients, allowing targeted therapies to be administrated with an improvement in overall survival. Nevertheless, these tumors develop mechanisms of resistance that are potentially targetable using novel therapies. Some molecular alterations can also modulate the treatment response. The molecular characterization of NS-NSCLC has to be performed in a short turnaround time (TAT), in less than 10 working days, as recommended by the international guidelines. In addition, the origin of the tissue biopsies for genomic analysis is diverse, and their size is continuously decreasing with the development of less invasive methods and protocols. Consequently, pathologists are being challenged to perform effective molecular technics while maintaining an efficient and rapid diagnosis strategy. Here, we describe the ultra-fast amplicon-based next-generation sequencing (NGS) workflow used in daily routine practice at diagnosis for NS-NSCLC patients. We showed that this system is able to identify the current molecular targets used in precision medicine in thoracic oncology in an appropriate TAT.

Author Spotlight: Advancements in Molecular Biomarker Testing for Non-Squamous Non-Small Cell Lung Cancer 

The increase of molecular biomarkers to be tested for non-squamous non-small cell lung cancer (NS-NSCLC) care management has prompted the development of fast and reliable molecular detection methods. We describe a workflow for genomic alteration assessment for NS-NSCLC patients using an ultra-fast-next generation sequencing (NGS) approach.

The number of molecular alterations to be tested for targeted therapy of non-squamous non-small cell lung cancer (NS-NSCLC) patients has significantly ...