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, a rapid viral RNA library preparation kit compatible with Illumina sequencers, provides an approximately five hour turn-around time from viral RNA sample to sequencing. The kit includes cDNA synthesis, SARS-CoV-2 enrichment, and library amplification and indexing reagents. The Enhanced protocol removes the need for normalization and quantification prior to amplification and indexing. RNA input is 5 μl, regardless of SC2 viral load in the sample extract. Amplification is performed using two primer pools that produce overlapping amplicons that are approximately 250 bp in size; as a result, no fragmentation is necessary prior to sequencing on Illumina platforms. 10 bp unique dual indexes (UDIs) 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.

Version Updates: V2-  Recommending 1:10 dilution of index primer on low viral titer samples to reduce short fragments. Controlled vocabulary for the use of positive control and negative template control has been added to the Sample Sheet following miseq loading guidelines. 

[STEPS]
SECTION: cDNA Synthesis Procedure
1. 
 

SECTION: cDNA Synthesis Procedure
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 Synthesis Procedure
3. While keeping reagents on ice, prepare the cDNA reaction according to the table below.  Briefly centrifuge, “gently yet thoroughly” vortex to mix, and centrifuge briefly again. For multiple reactions, prepare the calculated volume for all reactions, plus 10%. 

 

SECTION: cDNA Synthesis Procedure
4. 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: cDNA Synthesis Procedure
5. Proceed to“ SARS-CoV-2 Enrichment Procedure”. Alternatively, the samples can be stored at−30 to–15°C in a constant-temperature freezer.
6. 
 

SECTION: SARS-CoV-2 Enrichment Procedure
7. 
 

SECTION: SARS-CoV-2 Enrichment Procedure
8.  While keeping reagents on ice, set up Pool 1 and Pool 2 reactions for each sample, as described below. Briefly centrifuge, “gently yet thoroughly” vortex to mix, and centrifuge briefly again.
     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
9. 

Important: For samples with a broad/unknown range of Ct values (i.e., above and below Ct = 32) incubate as described in Table 9.1. For samples with Ct > 32, also incubate as described in Table 9.1. For samples with Ct value < 32, incubate as described in Table 9.2.

SECTION: SARS-CoV-2 Enrichment Procedure
9.1.   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: SARS-CoV-2 Enrichment Procedure
9.2.  Target enrichment cycling: 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
10. After amplification, combine the entire contents of “Pool 1” and “Pool 2” PCR reactions for each biological sample into a single well of a strip/plate, giving a volume of 50 µl.
SECTION: SARS-CoV-2 Enrichment Procedure
11. Add 50 µl QIAseq Beads to each 50 µl combined sample. Briefly centrifuge ,“gently yet thoroughly” vortex to mix, and centrifuge briefly again.


 

SECTION: SARS-CoV-2 Enrichment Procedure
12. Incubate for 5min at room temperature.
SECTION: SARS-CoV-2 Enrichment Procedure
13. Centrifuge the strip 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: SARS-CoV-2 Enrichment Procedure
14. With the strip 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: SARS-CoV-2 Enrichment Procedure
15. With the strip 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
16. Remove the strip 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
17. Centrifuge the strip 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
18. Transfer 28 μl to a clean plate. This is now “enriched SARS-CoV-2”.
SECTION: SARS-CoV-2 Enrichment Procedure
20. Important Notes: 


With wastewater samples anything that is 1ng/µl and above (even down to 0.5ng/µl with high Ct values) is a success and should definitely  move on to final amplification!
21. Proceed to “Library Amplification and Indexing Procedure”. Alternatively, the samples can be stored at –30 to –15°C in a constant-temperature freezer.
SECTION: Library Amplification and Indexing Procedure
22. 
 

SECTION: Library Amplification and Indexing Procedure
23. 
 

SECTION: Library Amplification and Indexing Procedure
24. For samples with a broad/unknown range of Ct values (i.e., above and below Ct = 32) incubate as described in Table 23.1. For samples with Ct > 32, also incubate as described in Table 23.1. For samples with Ct value < 32, incubate as described in Table 23.2.
SECTION: Library Amplification and Indexing Procedure
24.1. Library amplification/indexing cycling conditions: Samples with broad/unknown range Ct values and samples with Ct > 32


 

SECTION: Library Amplification and Indexing Procedure
24.2. Library amplification/indexing cycling conditions: Samples with Ct < 32


 

SECTION: Library Amplification and Indexing Procedure
25. On ice, prepare the library amplification and indexing reaction according to Table in Step 25. Briefly centrifuge, “gently yet thoroughly” vortex to mix, and centrifuge briefly again.


 

SECTION: Library Amplification and Indexing Procedure
26.  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
27. Transfer the strip to the thermal cycler and start the program.
SECTION: Library Amplification and Indexing Procedure
28. 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
29. Incubate the mixture for 5 min at room temperature.
SECTION: Library Amplification and Indexing Procedure
30. Centrifuge the strip until the beads are pelleted (2 min), and then place the strip 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
31. With the strip 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
32. With the strip 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
33. Remove the strip 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
34. Centrifuge the strip 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
35. Transfer 23 μl to a clean strip. 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
36. Quantify the library using Qubit HS assay kit. 
SECTION: Library Amplification and Indexing Procedure
37. Assess the quality of the library using a Bioanalyzer, Tape Station (D1000 tape), or Fragment Analyzer. Check for the expected size distribution of library fragments.
  



 

SECTION: Quantification, Normalization and Pooling of Libraries
38. 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 4 nM. Quantify the final pool at the end. 
  

SECTION: Quantification, Normalization and Pooling of Libraries
39.  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. Controlled vocabulary for labelling/naming positiveControl and negativeControl has been added to the Sample sheet.   

SECTION: SARS-CoV-2 Enrichment Procedure
19. Assess the enriched SARS CoV-2 using Tape station HS D5000 tape. Also, quantify using Qubit HS kit.