Protocol to decode the role of transcriptionally active microbes in SARS-CoV-2-positive patients using an RNA-seq-based approach

Summary The elucidation of the role of microorganisms in human infections has been hindered by difficulties using conventional culture-based techniques. Here, we present a protocol for the investigation of transcriptionally active microbes (TAMs) using an RNA sequencing (RNA-seq)-based approach. We describe the steps for RNA isolation, viral genome sequencing, RNA-seq library preparation, and metatranscriptomic and transcriptomic analysis. This protocol permits a comprehensive evaluation of TAMs’ contributions to the differential severity of infectious diseases, with a particular focus on diseases such as COVID-19. For complete details on the use and execution of this protocol, please refer to Devi et al.1


SUMMARY
The elucidation of the role of microorganisms in human infections has been hindered by difficulties using conventional culture-based techniques.Here, we present a protocol for the investigation of transcriptionally active microbes (TAMs) using an RNA sequencing (RNA-seq)-based approach.We describe the steps for RNA isolation, viral genome sequencing, RNA-seq library preparation, and metatranscriptomic and transcriptomic analysis.This protocol permits a comprehensive evaluation of TAMs' contributions to the differential severity of infectious diseases, with a particular focus on diseases such as COVID-19.For complete details on the use and execution of this protocol, please refer to Devi et al. 1

BEFORE YOU BEGIN
Comprehending the significance of TAMs in influencing the course or outcome of a disease represents a crucial aspect of clinical research that has not received in-depth examination thus far.This protocol outlines the precise steps required for conducting a functional metagenomics study among the various SARS-CoV-2 variants of concern (VOCs) from the nasopharyngeal swab isolated from the COVID-19 positive patients.These same procedures are applicable for investigating the metagenomic profile of any other sample/disease type.In case of blood samples, it is imperative to first eliminate globin mRNA along with the ribosomal RNA before proceeding with the preparation of the RNA-seq library.
Nasopharyngeal swab collection (performed by a trained healthcare provider, only) The trained paramedical staff at the MAX Hospital, Delhi collect the nasopharyngeal swabs of patients on the day of reporting to the hospital.Put the tip of the swab into a vial containing 3 mL of Viral Transport Media (VTM) (HiViral Transport Kit, HiMedia, Cat.No: MS2760A-50NO), by breaking the applicator's stick and sealing the tube tightly.Vortex the tube for 2 min to allow the dissolution of the sample into the VTM solution followed by centrifugation and allow it to settle for some time before processing.
Software and scripts used in this protocol are provided in the ''Software and algorithms'' section of the key resources table.
Note: A computer with a linux and network connection is required.The RAM requirement depends on the number of samples to be analyzed.16 GB RAM should be sufficient for an initial analysis.On the basis of availability of RAM, analysis time may fluctuate (fast).

STEP-BY-STEP METHOD DETAILS RNA isolation
Timing: 2 h This major step describes the isolation of RNA from the nasopharyngeal swab samples collected from hospital admitted COVID-19 positive patients.RNA is extracted using commercially available RNA extraction kits (QIAmp viral mini kit, Qiagen, Cat.No. 52906), in accordance with the kit protocol (QIAamp Viral RNA Mini Handbook).

Lyse the sample (VTM).
a. Take 200 mL of VTM solution in a 1.5 mL microcentrifuge tube.b.Add 560 mL prepared Buffer AVL containing carrier RNA.c. Mix thoroughly by pulse-vortexing for 15 s to ensure efficient lysis.d.Incubate at room temperature for 10 min.
CRITICAL: Buffer AVL-carrier RNA should be prepared fresh, and is stable at 2 C-8 C for up to 48 hours.This solution develops a precipitate when stored at 2 C-8 C that must be re-dissolved by warming at 80 C before use.
Note: Add Buffer AVE to the tube containing lyophilized carrier RNA to obtain a solution of 1 mg/mL (i.e., add 310 mL Buffer AVE to 310 mg lyophilized carrier RNA).Dissolve the carrier RNA thoroughly, divide it into conveniently sized aliquots, and store it at À20 C. Do not freeze-thaw the aliquots of carrier RNA more than 3 times.
2. Binding of the viral RNA to the QIAamp membrane.a. Add 560 mL 96% ethanol to the sample.b.Mix by pulse-vortexing for 15 s.c.Carefully apply 630 mL of the solution to the QIAamp Mini column.d.Close the cap, and centrifuge at 6000 3 g (8000 rpm) for 1 min.e. Place the QIAamp Mini column into a clean 2 mL collection tube, and discard the tube containing the filtrate.f.Repeat this step until all of the lysate has been loaded onto the spin column.a. Carefully open the QIAamp Mini column, and add 500 mL Buffer AW1. b.Close the cap and centrifuge at 6000 3 g (8000 rpm) for 1 min.c.Place the QIAamp Mini column in a clean 2 mL collection tube and discard the tube containing the filtrate.d.Carefully open the QIAamp Mini column and add 500 mL Buffer AW2.e. Close the cap and centrifuge at full speed (20,000 3 g; 14,000 rpm) for 3 min.
Note: Buffer AW1 and AW2 are supplied as a concentrate.Before using for the first time, add the appropriate amount of ethanol (96%-100%) to Buffer concentrates.Pause point: RNA collected can be stored at À80 C until library preparation.However, RNA is prone to degradation and samples should be used as soon as possible.To avoid possible batch effects due to different storage times, RNA from all study groups (Pre-VOCs, VOCs: Delta and Omicron) need to be included in each batch.

Timing: 2 h
We perform a real-time reverse transcription polymerase chain reaction (RT-PCR) test for SARS-CoV-2 detection and quantification using TRUPCR SARS-CoV-2 kit (3B BlackBio Biotech India Ltd.).8. Thaw the necessary kit (HiScribe T7 ARCA mRNA Kit (with tailing)) components, mix and pulsespin in microfuge to collect solutions to the bottoms of tubes.9. Reaction preparation.
a. Prepare the reaction mix as follows: a. Define the following setting for temperature profile.
11. Channel selection.a. Define the following setting for channel selection.
12. Result analysis: We consider the cycle threshold (Ct) value of 35 for interpretation of the results.

SARS-CoV-2 whole genome sequencing
Timing: 16 h COVID-19 positive RNAs are subjected to sequencing, which allows the identification of SARS-CoV-2 variants for further classification of samples into Pre-VOCs and VOCs (Delta and Omicron).
Note: Prepare the sequencing library following the recommendation of Oxford Nanopore Native Barcoding protocol.
13. Reverse transcription.a. Proceed with first strand cDNA generation from the isolated RNA using the following reaction mix.
b. Do pipette mixing and give a short spin.c.Put the reaction plate for incubation at 65 C for 5 min, and then snap cool on ice for 1 min followed by first strand cDNA synthesis.14.First strand cDNA synthesis.
a. Add the following reagents to the annealed template RNA.a. Bead Addition and Mixing: Add 45 mL of AMPure XP beads to the double-stranded cDNA and ensure thorough mixing by vortexing.b.Incubation: Incubate the mixture at room temperature for 15 min to allow binding of cDNA to the beads.c.Magnetic Separation: Place the plate on a magnetic stand for 5 min, allowing the beads to settle on the magnet's side.Carefully remove the clear supernatant without disturbing the beads.d.Ethanol Wash: Wash the bead-bound cDNA twice with 100 mL of 80% ethanol to remove impurities.Ensure that residual ethanol is removed by spinning the plate.e.Air Drying: Allow the plate to air dry, ensuring that all residual ethanol evaporates.f.Resuspension: Resuspend the bead-bound cDNA in 26 mL of resuspension buffer and incubate for 2 min at room temperature.g.Elution: Carefully elute 25 mL of purified double-stranded cDNA from the beads.h.Concentration Measurement: Measure the concentration of the purified cDNA product using a NanoDrop or similar instrument.XP beads to the tube.Incubate at room temperature for 10 min on a hula mixer.21.Magnetic Separation: Transfer the tube to a magnetic stand for 5 min.Carefully remove the clear supernatant once the beads have moved towards the magnet completely.22. Washing: Resuspend the pellet in 700 mL of Short Fragment Buffer (SFB).Return the tube to the magnet, and remove the supernatant.Repeat this step for a total of two washes.23.Ethanol Wash: Wash the pellet with 100 mL of 80% ethanol.24.Elution: Elute the pooled barcoded sample with 35 mL of nuclease-free water.25.Quantification: Quantify the pooled barcoded sample using the Qubit High Sensitivity dsDNA assay kit.26.Add the sequencing adapter to the pooled barcoded sample as per the following reaction: a. Gently pipette mix and spin the tube.b.Incubate the sample at room temperature for 20 min.27.Purification: Purify the final library using 20 mL AMPure XP beads.Following separation of beads from the library by placing onto the magnetic stand, wash the pellet twice using 125 mL Short Fragment Buffer (SFB) and elute with 15 mL Elution Buffer.28.Quantification: Quantify the final library using 1 mL of library in Qubit High sensitivity dsDNA assay kit.a.If concentration is above 100 ng/mL, dilute with EB buffer for a final concentration of 50 ng/mL in a volume of 13 mL.Measure the concentration again using 1 mL of sample in Qubit.29.Priming and loading the SpotON flow cell.
a. Allow the flow cell to come to room temperature ($ 24 C) for at least 5 min before inserting it into the MinION Mk1B device.b.Determine the number of active pores in the flow cell by selecting ''Check flow cell'' in MinKNOW.c. Open the priming port and check for a small air bubble under the cover.ii.Insert a P1000 tip into the priming port.
iii.Turn the pipette's thumbwheel anti-clockwise until the dial shows a gain of no more than 20-30 mL or until a small volume of storage buffer (yellow color) is entering the pipette tip.d.Prepare the flow cell priming mix by adding 30 mL of FLT to the entire tube (1,170 mL) of FB, and mix by pipetting, being careful to avoid foaming/air bubbles.e.The priming process involved two steps: i. First Priming -Open the priming port.Slowly, load a total of 800 mL of the priming solution into the flow cell.
Allow the flow cell to incubate at room temperature for 5 min.ii.Second Priming: For the second priming step, open the SpotON port as well.Add 200 mL of priming solution into the flow cell.These steps ensure that the MinION flow cell is properly primed and ready for sequencing.30.Sequencing run.
a.The final library was prepared for loading as per the formula mentioned below.
b. Mix the entire library very slowly by pipetting, followed by loading into the flow cell through the SpotON port in a dropwise manner.c.Configure the MinION Mk1c software for sequencing, selecting the high-accuracy base calling option.This configuration was done after providing the relevant information about the library preparation kit, including SQK-LSK109, EXP-NBD104, and EXP-NBD114.d.Allow the sequencing process to continue until a sufficient amount of data has been generated, and subsequently, stop the sequencing run.e.Once the sequencing run has been stopped, wash the flow cell using a Flow Cell wash kit, and QC it to confirm if there are still a sufficient number of functional pores on the flow cell for another sequencing run.f.Following this, store the flow cell at 4 C to preserve its integrity, and transfer the generated sequencing data to an HPC cluster for further data analysis.

Timing: 5-6 h
This step explains the detailed description of the analysis methods used for interpretation of the SARS-CoV-2 variant.
Note: It can copy locally, to/from another host over any remote shell, or to/from a remote rsync daemon.

Reagent Volume Storage
Sequencing Buffer (SQB) 37.5 mL À20 C Loading Beads (LB), mixed immediately before use 25.-scheme-directory: specifies the directory containing primer schemes for the analysis.
-read-file: specifies the location of the input FASTQ file containing the sequencing reads.
-fast5-directory: specifies the directory containing the raw FAST5 files generated by the sequencer.
-sequencing-summary: specifies the location of the sequencing summary file.This file contains information about the sequencing run, such as the number of reads generated, quality metrics.
''Sample_name'': specifies the name of the sample being analyzed.
Replace /path/to/ with the actual paths to the relevant directories and files on system.STAR Protocols This method explains how to convert total RNA into a library suitable for subsequent sequencing.
Steps involve RNA quantification, ribosomal RNA removal, RNA fragmentation, first and second cDNA strand synthesis, 3 0 end adenylation, adapter ligation, DNA fragment enrichment, final library quality and quantity assessment, cluster generation, and sequencing of reads 1 and 2.
44. RNA Quality Check.a. Check the concentration and purity of the isolated RNA using the Nanodrop system.b.For library preparation, take the input RNA volume according to the 250 ng concentration and make up the volume to 10 mL using nuclease-free water.45.Depletion of rRNA from total RNA.In this step, rRNA is depleted from each sample and further purified, fragmented and primed for cDNA synthesis.Bind rRNA a.In a 96-well plate, add an input concentration of 250 ng of the RNA and make up the volume to 10 mL for each sample using NFW.b.Add 5 mL RBB (rRNA Binding Buffer) to each well.c.Add 5 mL RRM G (rRNA Removal Mix -Gold) specific for the Ribo-zero gold kit to the well.d.Carefully mix the reagents by pipetting up and down 10 times (Do not vortex mix).e. Centrifuge at 280 g for 1 min.f.Incubate the reaction plate in the thermal cycler at 68 C for 5 min to allow denaturation of the RNA.g.Once the reaction is over, take the plate out and incubate at room temperature for 1 min.
Remove rRNA h.Thoroughly vortex and add 35 mL RRB (rRNA Removal Beads) to each well of the above plate.i.Again, mix by pipetting up and down 10 times.j.Incubate at room temperature for 1 min.k.Place the plate on a magnetic stand for 1 min.l.Transfer all the supernatant to the corresponding well of the 96-well plate.
Clean up RNA m.Vortex RNAClean XP Beads until well dispersed.n.Add 99 mL or 193 mL (if starting with degraded total RNA) RNAClean XP Beads to each well, and then mix thoroughly by pipetting up and down nearly 10 times.o.Incubate at room temperature for 15 min.p. Place on a magnetic stand and wait until the liquid is clear ($5 min).q.Remove and discard all of the supernatant from each well.r.Ethanol washing: i. Add 200 mL freshly prepared 70% EtOH to each well to wash.
ii. Incubate on the magnetic stand for 30 s.
iii.Remove and discard all supernatant from each well.(Remove all the residual EtOH from each well).iv.Air-dry on the magnetic stand for 15 min.v. Remove from the magnetic stand.
Note: Always use freshly prepared Ethanol.Make sure all the drops of ethanol are removed/ dried from the walls of the plate before adding ELB.

s. Elution:
i. Add 11 mL ELB to each well, and then mix thoroughly by pipetting up and down.
ii. Incubate at room temperature for 2 min.
iii.Centrifuge at 280 3 g for 1 min.iv.Place on a magnetic stand and wait until the liquid is clear ($5 min).v. Transfer 8.5 mL supernatant to the corresponding well.t.Add 8.5 mL EPH to each well, and then mix thoroughly by pipetting up and down.u.Incubate on the thermal cycler at 94 C for 8 min and hold at 4 C. v. Centrifuge briefly after taking out of the thermal cycler.
CRITICAL: Always avoid mixing using a vortexer during the complete library preparation protocol (do pipette mixing).All the beads must be taken out and let stand at room temperature for 30 min (never use cold beads).Especially, rRNA Removal Beads are highly dense, requiring meticulous and deliberate pipetting for precise and accurate handling.During cleanup, transfer the supernatant carefully by checking under a white light or on a white surface for the beads on tips.

First strand cDNA synthesis.
In this step, the cleaved RNA fragments are reverse transcribed into the first strand cDNA.Check the concentration and purity of the isolated RNA using the Nanodrop system.a. Add SuperScript IV at a ratio of 1 mL SuperScript IV to 9 mL FSA.
Note: SuperScript IV is an enzyme and should be taken out from  Note: Expect the final product to be a band at $280 bp.A single peak should be observed for the library.If an unexpected small peak is observed at 120-170 bp, it indicates the presence of adapter dimers (Figure 2A).If adapter dimers are present in the library, perform an additional clean-up step with AMPure beads (second round of purification may reduce the library yields).
A bead ratio of 0.8x to 1x is usually recommended and sufficient to remove the unwanted adapter dimers (Figure 2B).
CRITICAL: One day prior to anticipated run, remove cartridge from À20 C storage.Thaw at room temperature for 6 hours.Transfer to refrigerator 4 C, and continue to thaw for a minimum of 12 hours for 300 cycle or smaller kits.Before beginning with normalization and pooling, thaw the cartridge at room temperature, cartridge should have air on all sides except the bottom and should not be stacked.The NextSeq flow cell should also be taken out from 4 C at this point (half an hour before final loading of the library)

Normalize and Pool Libraries.
Note: For best practice, perform normalization and pooling directly prior to sequencing.To minimize index hopping, do not store libraries in the pooled form.# Here, the command ''hisat2-build'' is used to create index of human reference sequence.
# hisat2 was used to map the query sequence from human reference sequence.Since, hisat gives output as .samfile, samtools is used to sort and convert the output in .bamfile using the code below.ii.Download database here.https://lomanlab.github.io/mockcommunity/mc_databases.html.iii.Download the database from the Kraken2 website, which contains bacteria, archaea, and viral reference sequences.iv.Kraken2 map reads with taxa using k-mers from the genomic database and assigns taxonomy.v. Run: vi.Output: Note: Although, kraken2 provides faster and more accurate classification, however, it cannot assign each read to the species.To overcome this limitation, we analyzed Kraken2 output with Bracken2.The provided screenshot illustrates the increase in reads identified by Bracken2 compared to those assigned by Kraken2 (reads assigned to a specific species in a particular sample).

Transcriptome analysis
Timing: 1-2 days (according to the availability of computational resources, may take less time) 63. Indexing Transcriptome and Quantifying Reads using Salmon.a. Download reference transcriptome in FASTA format for the Protein-coding transcript sequences on the human reference from (https://www.gencodegenes.org/human/).
Note: This will save the differential expression results to a comma-separated file (csv) file for further analysis or visualization.The analysis outcome will produce a CSV file that presents the outcomes of the differential expression tests for all genes, ordered by the adjusted p-value.The columns in the file will encompass the mean expression, fold change between conditions, relevant statistical test metrics, p-value and adjusted p-value.The adjusted p-values are derived from the p-values and should be used in preference over the p-values.Use this analysis to identify the correlation between bacterial species and the expressed host genes.

Pathway enrichment analysis
66. Utilize microbial species for correlation analysis with differentially expressed host genes using the corrr (0.4.4) package in R, employing Spearman's correlation analysis.67.Establish a correlation coefficient cut-off of RG0.8 and a p-value of %0.05 to construct a correlation plot and visualize the interaction network in Cytoscape (version 3.9.1), a GUI-based tool.

EXPECTED OUTCOMES
The major outcome of this protocol involves the identification of Transcriptionally Active Microbes (TAMs) and their integration with host-response genes.Through an integrative approach of dual RNA-Sequencing and metagenomic analysis, this method provides new insights into potentially functional host-microbiome dynamics and explores the association between host pathways, genes, and functional microbial species.This method offers an effective means of identifying putative microbial candidates' vis-a `-vis various diseases caused by a primary infecting pathogen.

LIMITATIONS
The RNA-seq protocol has been fine-tuned to work best with a substantial input of total RNA, typically ranging from 0.1 to 1 mg.This range offers an ideal opportunity to include a diverse range of RNA samples.However, it's crucial to optimize the protocol initially to ensure that each sample is consistently processed with the same RNA concentration.This approach eliminates any potential bias in further steps of library preparation.This is even more important as we undertake integrative analysis for the expressed host genes and the transcribed microbes in those cohorts of patients.
Moreover, one of the protocol's key steps involves removing rRNA to enrich the desired mRNA reads.This step significantly increases the coverage of the desired mRNA transcripts.Unfortunately, it's observed that not every sample in our library preparation process achieves uniform rRNA depletion.This discrepancy results in some more reads being assigned to rRNA in few samples.This protocol focuses mainly on the coding mRNA and long non coding RNAs leaving the small mRNAs.
Additionally, during the purification step of adapter ligation, if not executed meticulously, there's a risk of adapter dimer formation.These adapter dimers can be problematic for the success of the library preparation process.If not adequately removed, they may lead to issues in our final library.

Potential solution
Make sure that the rRNA removal beads (RRB) are at room temperature before adding it to the sample and do not allow the RRB pellets to dry.Pipette up and down quickly to ensure thorough mixing.Insufficient mixing leads to lower levels of rRNA depletion.Pipette with the tips at the bottom of the well to prevent foaming.Excess foam leads to sample loss because foam does not transfer efficiently.

Potential solution
The fragmentation of nucleic acids is a crucial step in optimizing library preparation, clustering, and sequencing, and the specific conditions for fragmentation depend on the initial state of the RNA, whether it's intact or degraded.When working with intact RNA, the TruSeq Stranded Total RNA Library Prep protocol for transcriptome analysis involves fragmenting the RNA after rRNA depletion.For samples containing degraded RNA, it's important to avoid over-fragmentation.In such cases, the fragmentation time should be reduced.This can be achieved by either omitting or modifying the thermal cycler Elution 2-Frag-Prime program to involve a 94 C incubation for X min, followed by a 4 C hold.

Potential solution
A Bioanalyzer chip is run to check the adapter dimers in the library which hampers the library binding to the flow cell.So to remove dimers before putting sequencing is inevitable and for this 1:1 AMPure beads purification is done for libraries which have adapter dimers in BA profile.

Problem 4
Storage issue during HUMAnN3 analysis (related to Step 62).

Protocol
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
CRITICAL: Use only ethanol, since other alcohols may result in reduced RNA yield and purity.Only use freshly prepared ethanol.STAR Protocols 5, 103071, June 21, 2024 Protocol 3. Wash.

4 .
Dry Spin.a. Place the QIAamp Mini column in a new 2 mL collection tube, and discard the old collection tube with the filtrate.b.Centrifuge at full speed for 1 min.CRITICAL: Dry spin is always recommended since residual Buffer AW2 in the eluate may cause problems in downstream applications.5. Elute.a. Place the QIAamp Mini column in a clean 1.5 mL microcentrifuge tube.Discard the old collection tube containing the filtrate.b.Carefully open the QIAamp Mini column and add 60 mL Buffer AVE equilibrated to room temperature.c.Close the cap and incubate at room temperature for 1 min.d.Centrifuge at 6000 3 g (8000 rpm) for 1 min.6. Quantify the isolated RNA using a NanoDrop Spectrophotometer taking 1 mL of the sample.7. Store the viral RNA at À20 C or À80 C for long term storage.
back a small volume to remove any bubble:
two times with 200 mL fresh 80% EtOH as follows.ix.Incubate on the magnetic stand for 30 s.x.Remove and discard all supernatant from each well.xi.Air-dry on the magnetic stand for 15 min.xii.Remove from the magnetic stand and add 32.5 mL RSB to each well, and then mix thoroughly by pipetting up and down 10 times.xiii.Incubate at room temperature for 2 min.xiv.Centrifuge at 280 3 g for 1 min.xv.Place on a magnetic stand and wait until the liquid is clear (2-5 min).xvi.Transfer 30 mL supernatant to the corresponding well of the TSP1 plate.Pause point: If you are stopping, seal the plate and store at À25 C to À15 C for up to 7 days.51.Quantify Libraries.a. Use 1 mL sample to quantify all the libraries individually using the Qubit dsDNA HS Assay kit.b.Check the size of the quantified libraries using a DNA 1000 chip on an Agilent Technologies 2100 Bioanalyzer using Bioanalyzer High Sensitivity DNA kit.

Figure 2 .
Figure 2. BioAnalyzer profile for the prepared library (A) 2 peaks indicating the adapter dimer (140 bp) and library (280 bp).(B) After cleanup with beads, adapter dimer removed and only 1 peak indicating purified library.
Mix using pipette and spin down the residual volume.c.Put the reaction plate in the thermal cycler for incubation as per using the below program.
-STAR Protocols 5, 103071, June 21, 2024 Protocol b. d.To degrade the RNA strand attached to the cDNA, add 1 mL of RNase H to each well.e. Mix properly and incubate at 37 C for 20 min.15.Second strand cDNA synthesis.a. Prepare the following master mix and add it to cDNA.b.Mix by vortexing, spin down the residual volume.c.Incubate the plate at 95 C for 3 min and snap cooled on ice for >1 min.d.Add 1 mL of Klenow Fragment to each well, followed by vortexing and spinning down the residual volume.e. Incubate the plate in a thermal cycler as per the program below.16.Double stranded cDNA purification.
. Operating System Requirement: A 64-bit UNIX, Linux, or a similar environment.This includes Mac OS, Linux distributions such as Ubuntu 16 or later, or Windows 10 Subsystem for Linux.b.Create a custom conda environment: Install the relevant version of Miniconda (recommended as it is relatively small and quick to install) following the installation guidelines at www.conda.io/projects/conda/en/latest/user-guide/install/index.html.c.Install ARTIC nCoV-2019 specific data and software repository: Update the environment using the following command.
5, 103071, June 21, 2024 40.Convert the barcode name with Sample name: Perform this step for individual barcodes.41.Change of SARS-CoV-2 header with Institute tag name for all the samples 42.Concatenate all fasta to one file.fori in *.fasta;do temp = ${i%.*};echo"sed'1s/>MN908947.3/>${temp}_CSIR-IGIB/g'$i> ./fastachanged/$temp.fasta";done> name.shbashname.sh.#An alternative way to change of SARS-CoV-2 header with sample tag name for each samples individually.sed'1s/>MN908947.3/>${temp}_CSIR-IGIB/g'barcode01.fasta>./fastachanged/barcode01.consensus.fastased'1s/>MN908947.3/>${temp}_CSIR-IGIB/g'barcode02.fasta>./fastachanged/barcode02.consensus.fastased'1s/>MN908947.3/>${temp}_CSIR-IGIB/g'barcode03.fasta>./fastachanged/barcode03.consensus.fasta.forfilename in barcode01*; do echo mv \"$filename\" \"${filename//barcode01/IGIB1130001}\"; done | /bin/bash.# Here, ''| /bin/bash/'' pipes the printed 'mv' commands into the Bash shell (/bin/bash) for execution.# An alternative approach using 'rename' command for converting barcode names to sample names for each sample.rename -v 's/barcode01/IGIB1130001/' * cat *.consensus.fasta> concatenated_consensus.fasta.Protocol Timing: $ 50 min with handling time (for step 46) Timing: $ 2 h with handling time (for step 47) Timing: $ 45 min with handling time (for step 48) Timing: $2 h 30 min with handling time (for step 49) À20 C temperature at the time of use only, and in a cooler/ ice.FSA should be handled with precautions since it contains Actinomycin D, a toxin b.Add 8 mL FSA and SuperScript II mixture to each well of the plate, and then mix thoroughly by pipetting up and down.c.Centrifuge at 280 3 g for 1 min.d.Incubate on the preprogrammed thermal cycler and run the following ''synthesize 1st Strand program''.Each well contains 25 mL..Place on the bench and let stand to bring to room temperature.i.Purify cDNA.i.Add 90 mL AMPure XP beads to each well of the DFP plate.ii.Mix thoroughly by pipetting up and down 10 times.iii.Incubate at room temperature for 15 min.iv.Centrifuge at 280 3 g for 1 min.v.Place on a magnetic stand and wait until the liquid is clear ($5 min).Protocol v. Place on a magnetic stand and wait until the liquid is clear ($5 min).vi.Transfer 15 mL supernatant to the corresponding well of the plate.Incubate at 30 C for 10 min on the thermal cycler.g.Add 5 mL STL to each well, and then pipette mix thoroughly.h.Centrifuge at 280 3 g for 1 min.i.CleanUp Ligated Fragments: This cleanup is done in 2 rounds with different volumes of AM-Pure XP beads and RSB.Perform steps i. to xvii using the Round 1 volumes.Repeat steps i. through xvii with the new plate using the Round 2 volumes.If you are stopping, seal the plate and store at À25 C to À15 C for up to 7 days.50.Enrich DNA Fragments.This process uses PCR to selectively enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library.PCR is performed with PPC (PCR Primer Cocktail) that anneals to the ends of the adapters.Minimize the number of PCR cycles to avoid skewing the representation of the library.a.Amplify DNA Fragments.i.Place the PCR plate on ice and add 5 mL PPC to each well.ii.Add 25 mL PMM to each well, and then mix thoroughly by pipetting up and down 10 times.iii.Centrifuge at 280 3 g for 1 min.iv.Place on the preprogrammed thermal cycler and run the PCR program.Each well contains 50 mL.v. Choose the preheat lid option and set to 100 C. Mix thoroughly by pipetting up and down 10 times.iv.Incubate at room temperature for 15 min.v. Centrifuge at 280 3 g for 1 min.vi.Place on a magnetic stand and wait until the liquid is clear (2-5 min).vii.Remove and discard all supernatant from each well.
47. Second strand cDNA synthesis.This process removes the RNA template, synthesizes a replacement strand, and incorporates dUTP in place of dTTP to generate ds cDNA.The incorporation of dUTP quenches the second strand during amplification.Magnetic beads separate the ds cDNA from the second strand reaction mix.The result is blunt-ended cDNA.a.Dilute CTE to 1:50 in RSB.For example, 2 mL CTE + 98 mL RSB.b.Add 5 mL diluted CTE to each well.Discard diluted CTE after use.c.Add 5 mL RSB to each well.d.Centrifuge SMM at 600 3 g for 5 s.e.Add 20 mL SMM to each well, and then mix thoroughly by pipetting up and down 6 times.f.Centrifuge at 280 3 g for 1 min.g.Place on the preprogrammed thermal cycler and incubate at 16 C for 1 h.Each well contains 50 mL.hv.Air-dry on the magnetic stand for 15 min.Do not over dry beads.k.Elution.i.Remove from the magnetic stand.ii.Add 17.5 mL RSB to each well, and then pipette mix thoroughly.iii.Incubate at room temperature for 2 min.iv.Centrifuge at 280 3 g for 1 min.d.Add 12.5 mL ATL to each well, and then mix thoroughly by pipetting up and down 10 times.e.Seal the ALP plate with a Microseal 'B' adhesive seal.f.Centrifuge at 280 3 g for 1 min.g.Put the samples on the thermal cycler and initiate the ATAIL70 program.Utilize the preheat lid option, setting it to 100 C, and then proceed with incubation at 37 C for 30 min, followed by 70 C for 5 min.Hold at 4 C.Each well contains 30 mL. h.Centrifuge at 280 3 g for 1 min.49.Ligate Adapters.This process ligates multiple indexing adapters to the ends of the ds cDNA fragments, which prepares them for hybridization onto a flow cell.a. Dilute CTL 1:100 in RSB.For example, 1 mL CTL + 99 mL RSB.Discard the diluted CTL iii.Incubate at room temperature for 15 min.iv.Centrifuge at 280 3 g for 1 min.v. Place on a magnetic stand and wait until the liquid is clear (2-5 min).vi.Remove and discard all supernatant from each well.vii.Wash two times with 200 mL fresh 80% EtOH by incubating on the magnetic stand for 30 s.