TITLE: Enhanced Qiaseq Direct SARS-CoV-2 kit  for Illumina Miseq
AUTHORS: Padmini   Ramachandran, Tamara  Walsky, Amanda  Windsor, Maria  Hoffmann, Chris   Grim

[DESCRIPTION]
This SARS-CoV-2 targeted, and rapid viral RNA library preparation and sequencing kit provides an approximately five hour turn-around time from viral RNA sample to sequencing.The kit includes cDNA synthesis, enrichment, and library amplification and indexing.The enhanced protocol removes the need for normalization and quantification prior to amplification and indexing.RNA input is 5 ul, regardless of SC2 viral load in the sample extract.Amplification is performed using two primer pools that produces amplicons that are 300 bp in size, so no fragmentation is necessary prior to sequencing on Illumina platforms.10 bp unique dual indices are provided in the kit, and with a cycle read length of 149 for each paired-end read, the sequencing time is just over 28 hours.

[STEPS]
SECTION: cDNA setup
1. 
 

SECTION: cDNA setup
2. Thaw template RNA on ice. Gently yet thoroughly mix, then briefly centrifuge to collect residual liquid from the sides of the tubes and return to ice.
SECTION: cDNA setup
3. Thaw RP Primer (random hexamer), Multimodal RT Buffer, and nuclease-free water at room temperature (15–25°C).
SECTION: cDNA setup
4. Mix thoroughly and then briefly centrifuge to collect residual liquid from the sides of the tubes. Prolonged storage of Multimodal RT Buffer at -20°C can cause white precipitate to form. This is normal. Just ensure to mix until precipitate is fully dissolved.
SECTION: cDNA setup
5. Thaw RNase Inhibitor and EZ Reverse Transcriptase on ice. Mix by flicking the tubes, briefly centrifuge, and return to ice. After use, immediately return the enzymes to the freezer.
SECTION: cDNA setup
6. While keeping reagents on ice, prepare the cDNA reaction according to the table below.  For multiple reactions, prepare the calculated volume for all reactions, plus 10% 
 

SECTION: cDNA setup
8. Proceed to“ SARS-CoV-2 Enrichment Procedure”. Alternatively, the samples can be stored at−30 to–15°C in a constant-temperature freeze.
9. 
 

SECTION: SARS-CoV-2 Enrichment Procedure
10. 
 

SECTION: cDNA setup
7. cDNA synthesis incubation.  Incubate as described below.
 
     Step      Temperature (°C)      Incubation time        1      25      10 min        2      42      50 min        3      85      5 min        4      4      Hold   
 
SECTION: SARS-CoV-2 Enrichment Procedure
11.  While keeping reagents on ice, set up Pool 1 and Pool 2 reactions for each sample, as described below
     Component      Pool 1: Volume/reaction (µl)      Pool 2: Volume/reaction (µl)        cDNA from “Protocol: Enhanced QIAseq DIRECT”      8      8        DIRECT SARS-CoV-2 Pool  1      2      ---        DIRECT SARS-CoV-2 Pool 2      ---      2        UPCR Buffer, 5x      5      5        QN Taq Polymerase      1      1        Nuclease-free water      9      9        Total volume      25      25   
 
SECTION: SARS-CoV-2 Enrichment Procedure
12. 
 


SECTION: SARS-CoV-2 Enrichment Procedure
13. After amplification, combine the entire contents of “Pool1” and “Pool2” PCR reactions for each biological sample into a single well of a plate, giving a volume of 50µl
SECTION: SARS-CoV-2 Enrichment Procedure
14. Add 50µl QIAseq Beads to each 50µl combined sample. Briefly centrifuge ,“gently yet thoroughly” vortex to mix, and centrifuge briefly again.
SECTION: SARS-CoV-2 Enrichment Procedure
15. 
 

SECTION: SARS-CoV-2 Enrichment Procedure
16. Incubate for 5min at room temperature
SECTION: SARS-CoV-2 Enrichment Procedure
17. Centrifuge the plate until the beads are pelleted(2min), and then place the plate onto a magnetic rack. After the solution has cleared(2min or longer), carefully remove and discard the supernatant.

  

SECTION: Library Amplification and Indexing procedure
27. 
 

SECTION: Library Amplification and Indexing procedure
28. Prepare the reagents required for library amplification and indexing.

Thaw the QIAseq DIRECT UDI Index Set A, B, C, or D, as well as the UPCR Buffer, 5x, at room temperature. “Gently yet thoroughly” vortex to mix, and then centrifuge briefly.

Thaw the QN Taq Polymerase. Mix by flicking the tubes, and then centrifuge briefly.
SECTION: Library Amplification and Indexing procedure
30. On ice, prepare the library amplification and indexing reaction according to Table in Step 31. Briefly centrifuge, “gently yet thoroughly” vortex to mix, and centrifuge briefly again.

Note: If setting up more than one reaction, prepare a volume of master mix 10% greater than what is required for the total number of reactions
SECTION: SARS-CoV-2 Enrichment Procedure
12.2.  Target enrichment cycling: Samples with broad/unknown range Ct values and samples with Ct > 32
      Step  Time  Temperature (°C)  Number of cycles    Heat activation  2   min  98  1      Denaturation  20 s  98  4    Annealing/extension  5 min  63      Denaturation  20 s  98  20    Annealing/extension  3 min  63    Hold  ∞  4  Hold  
 
SECTION: SARS-CoV-2 Enrichment Procedure
12.1.   Target enrichment cycling: Samples with broad/unknown range Ct values and samples with Ct < 32
      Step  Time  Temperature (°C)  Number of cycles    Heat activation  2   min  98  1      Denaturation  20 s  98  4    Annealing/extension  5 min  63      Denaturation  20 s  98  29    Annealing/extension  3 min  63    Hold  ∞  4  Hold  
 
SECTION: Library Amplification and Indexing procedure
31.  Reaction mix for library amplification and indexing
        Component  Volume/reaction    “Enriched   SARS-CoV-2” sample  24 μl    Index from   QIAseq DIRECT UDI index plate (A, B, C, or D) Plate  2 μl    Nuclease-free   water  12 μl    UPCR Buffer 5x  10 μl    QN Taq   Polymerase  2 μl    Total reaction   volume  50 μl  
 
SECTION: Library Amplification and Indexing procedure
32. Transfer the plate to the thermal cycler and start the program.
SECTION: Library Amplification and Indexing procedure
33. Once PCR is complete, add 40 μl of resuspended QIAseq Beads to each 50 μl reaction. Briefly centrifuge, “gently yet thoroughly” vortex to mix, and centrifuge briefly again.
SECTION: Library Amplification and Indexing procedure
34. 
 

SECTION: Library Amplification and Indexing procedure
35. Incubate the mixture for 5 min at room temperature.
SECTION: Library Amplification and Indexing procedure
36. Centrifuge the plate until the beads are pelleted (2 min), and then place the plate onto a magnetic rack. After the solution has cleared (2 min or longer), carefully remove and discard the supernatant.
 

SECTION: Library Amplification and Indexing procedure
37. With the plate still on the magnetic stand, add 200 μl of 80% ethanol. Wait 1 min and carefully remove and discard the wash. Repeat the wash, for a total of 2 ethanol washes. Remove excess ethanol as much as possible.
 

SECTION: Library Amplification and Indexing procedure
38. With the plate still on the magnetic stand, air-dry at room temperature for 4–8 min (until the beads start to crack and pellet loses its shine).

  

SECTION: Library Amplification and Indexing procedure
39. Remove the plate from the magnetic stand and elute the DNA from the beads by adding 25 μl nuclease-free water. Vigorously vortex (triturate if necessary) to mix, briefly centrifuge, and incubate for 2 min.
SECTION: Library Amplification and Indexing procedure
40. Centrifuge the plate until the beads are pelleted (2 min), and then return the plate to the magnetic rack until the solution has cleared (2 min or longer).
SECTION: Library Amplification and Indexing procedure
41. Transfer 23 μl to a clean plate. This is the “SARS-CoV-2 library”. If not proceeding immediately, the sample can be stored at –30 to –15°C.
SECTION: Library Amplification and Indexing procedure
43. Assess the quality of the library using a Bioanalyzer, TapeStation (D1000 tape), or Fragment Analyzer. Check for the expected size distribution of library fragments. An example library is shown here.

  

SECTION: Quantification, Normalization and Pooling of Libraries
44. Using the attached Post library worksheet, calculate the nM based on the Qubit concentration (ng/ul) and the Tape station (bp) profile. Normalize the “SARS-CoV-2 library”, to 4nM. Quantify the final pool at the end. 
  

SECTION: Quantification, Normalization and Pooling of Libraries
45.  Proceed to loading on to MiSeq (150x 150 cycle) using the attached Sample Sheet. Alternatively, the purified “SARS-CoV-2 library” can be safely stored at –30 to –15°C in a constant-temperature freezer until ready to use for sequencing.  

SECTION: SARS-CoV-2 Enrichment Procedure
26. Proceed to “Library Amplification and Indexing Procedure”. Alternatively, the samples can be stored at –30 to –15°C in a constant-temperature freezer.
SECTION: SARS-CoV-2 Enrichment Procedure
25. 
 

SECTION: SARS-CoV-2 Enrichment Procedure
24. Transfer 28 μl to a clean plate. This is now “enriched SARS-CoV-2”.
SECTION: SARS-CoV-2 Enrichment Procedure
23. Centrifuge the plate until the beads are pelleted (2 min), and then return the plate to the magnetic rack until the solution has cleared.
SECTION: SARS-CoV-2 Enrichment Procedure
22. Remove the plate from the magnetic stand and elute the DNA from the beads by adding 30 μl nuclease-free water. “Gently yet thoroughly” vortex (triturate if necessary) to mix, briefly centrifuge, incubate for 2 min.
SECTION: SARS-CoV-2 Enrichment Procedure
21. With the plate still on the magnetic stand, air-dry at room temperature for 4–8 min (until the beads start to crack and pellet loses its shine).

  

SECTION: SARS-CoV-2 Enrichment Procedure
20. 
 

SECTION: SARS-CoV-2 Enrichment Procedure
19. With the plate still on the magnetic stand, add 200µl of 80% ethanol. Wait 1min and carefully remove and discard the wash. Repeat the wash, for a total of 2 ethanol washes. Remove excess ethanol as much as possible.
SECTION: SARS-CoV-2 Enrichment Procedure
18. Important: Do not discard the beads, as they contain the DNA of interest.

Tip: It may be valuable to discard the supernatant twice. The contents settle after the first discard.
SECTION: Library Amplification and Indexing procedure
29.2. Library amplification/indexing cycling conditions: Samples with Ct < 32


 

SECTION: Library Amplification and Indexing procedure
29.1. Library amplification/indexing cycling conditions: Samples with broad/unknown range Ct values and samples with Ct > 32


 

SECTION: Library Amplification and Indexing procedure
29. For samples with a broad/unknown range of Ct values (i.e., above and below Ct = 32) incubate as described in step 29.1. For samples with Ct > 32, also incubate as described in Table 29.1. For samples with Ct value < 32, incubate as described in step 29.2
SECTION: Library Amplification and Indexing procedure
42. Quantify the library using Qubit HS assay kit.