TITLE: SHIMS 3.0: Highly efficient single-haplotype iterative mapping and sequencing using ultra-long nanopore reads
AUTHORS: Daniel W  Bellott, Ting-Jan  Cho, Emily       K Jackson, Helen       Skaletsky, Jennifer F. Hughes, David       C Page

[DESCRIPTION]
The reference sequence of structurally complex regions can only be obtained through a highly accurate clone-based approach that we call Single-Haplotype Iterative Mapping and Sequencing (SHIMS). In recent years, improvements to SHIMS have reduced the cost and time required by two orders of magnitude, but internally repetitive clones still require extensive manual effort to transform draft assemblies into reference-quality finished sequences. Here we describe SHIMS 3.0, using ultra-long nanopore reads to augment the Illumina data from SHIMS 2.0 assemblies and resolve internally repetitive structures. This greatly minimizes the need for manual finishing of Illumina-based draft assemblies, allowing a small team with no prior finishing experience to sequence challenging targets with high accuracy. This protocol proceeds from clone-picking to finished assemblies in 2 weeks for about 80 dollars per clone. We recently used this protocol to produce reference sequence of structurally complex palindromes on chimpanzee and rhesus macaque X chromosomes. Our protocol provides access to structurally complex regions that would otherwise be inaccessible from whole-genome shotgun data or require an impractical amount of manual effort to generate an accurate assembly.

[BEFORE_START]
Prepare stock solutions described in Materials:
- 1M Tris-Cl, pH 8.5
- 10 mM Tris-Cl, pH 8.5 
- PEG buffer (18% PEG(wt/vol)/1M NaCl Solution)
- 2X LB
- Chloramphenicol
- 80% (vol/vol) Glycerol solution

Figure 1 provides an overview of the protocol.
Troubleshooting advice can be found in Table 1.

[GUIDELINES]
 
TIMING

Steps 1-3, pick clones and grow cultures: 1080 min
Steps 4-7, glycerol stock plate: 30 min
Steps 8-10, pooling clones: 60 min-120 min
Steps 11-33, alkaline lysis: 60 min-120 min
Steps 34-42, MinION library prep: 30 min
Steps 43-51, MinION library loading: 30 min
Steps 52-55, demultiplex reads: 30 min
Steps 56-58, Identify full-length reads: 30 min
Steps 59-65, generate consensus sequence: 30 min
Steps 66-71, finishing:  - 480 min



          Step               Problem               Possible Reason               Solution               33         Low DNA concentration          Culture undergrowth or   overgrowth         Check culture OD600 is between   0.2-0.35            Incomplete Lysis         Make sure to thoroughly mix the   solution until the color is uniform            Incomplete neutralization         Solution from step 13   should not appear viscous and precipitate should float to the surface            Incomplete DNA elution         Pre-warm elution buffer   to 50°C            34         Concentration varies when checking with NanoDrop or Qubit         DNA is not completely   mixed         After adjusting concentration from step 33, leave DNA solution   on a heated shaker   at the gentlest setting at 50°C until DNA is completely mixed            51         Pores decrease rapidly         Impure DNA sample         Re-check DNA concentration.   Extract DNA again if NanoDrop and Qubit results are discordant, 260/280  < 1.7, 260/280 > 2.0, 260/230 <   2.0,  or 260/230 > 2.2            Bubbles introduced during   loading         Pipet very slowly and take care   not to introduce bubbles during flow cell priming and library loading            55         No reads for one or more clones         Clone culture failed         Regrow and add to the next run, or   replace the clone with another            Regrow the clone for an additional   round of sequencing            Bookkeeping error; some common   bookkeeping errors result from transposing digits, rotating a plate by 180°,   or contamination from a clone in an adjacent well         Resolve bookkeeping error, and rerun   a new clone or replace with another clone                58         Low fraction of long reads         FRA treatment time too   long         Promptly heat-inactivate FRA at   35 seconds            Adjust the FRA incubation time   below 35 seconds            Shearing during library   prep         Use wide-bore tips for   all mixing and loading steps            71         Clone sequence is shorter than expected   or missing known sequence             Deletion during culture         Regrow the clone from the   original culture or another library copy, and replace with the alternate   clone            Sequence toxic to E. coli         Close the gap by   long-range PCR or region-specific extraction      
 
Anticipated Results

We typically pool 24 clones for a single MinION run, generating about 300,000 reads with a read n50 of 20 kb, and a total of about 1.5 Gb of sequence data. Each clone typically receives 1-5% of the total reads. Occasionally some clones will have no reads; this usually indicates that the culture of the clone (Steps 1-3) has failed (see troubleshooting information for Step 55).

Expect to obtain 3-10 full-length reads per clone. Because of the high rate of insertions and deletions in individual nanopore reads, full-length reads may differ in length by 10 kb or more. Occasionally, a clone will have no reads that start and end in vector sequence, but the clone length will be apparent from a peak in the tail of the distribution of read lengths. It may still be possible to reconstruct a full-length consensus sequence by rotating one of these putative full-length reads to place the vector sequence at the beginning. However, we do not recommend this procedure for internally repetitive clones, particularly tandem arrays. Instead, sequence the clone again, and use these ambiguous reads to help polish the consensus.

[STEPS]
SECTION: Pick Clones and Grow Cultures
1. Fill each well of a Nunc 96 DeepWell plate with 1.9 mLof 2X LB containing 34 µg/ml  chloramphenicol.
 

SECTION: Pick Clones and Grow Cultures
2. Use a clean pipette tip to scrape the surface of a frozen glycerol stock and drop the tip directly into the DeepWell plate to inoculate a well. Inoculate each sample 8 times for a total of 15.2 mL per sample (8 x 1.9 mL). 24 samples in total for each library prep.

 

SECTION: Pick Clones and Grow Cultures
3. Seal plates with AirPore Tape Sheets and incubate on a shaker at   .

 

SECTION: Glycerol Stock Plate
4. Dispense 150 µL of 80% (vol/vol) glycerol solution into two rows of a Costar Assay Plate.

SECTION: Glycerol Stock Plate
5. Transfer 150 µL of each sample culture from Step 3 to a corresponding well of the assay plate and mix by pipetting up and down 20 times.

SECTION: Glycerol Stock Plate
6. Seal the glycerol stock plate with aluminum adhesive foil.
SECTION: Glycerol Stock Plate
7. Store the glycerol stock plate at -80 °C 

SECTION: Pooling Clones
8. Pour overnight cultures from Step 3 into a large beaker to combine pool.
SECTION: Pooling Clones
9. Divide pooled culture into two 250 mL Nalgene bottles and spin down culture at 6000 x g, 30 min, 4 °C 
SECTION: Pooling Clones
10. Remove media by pouring into a waste-collecting container. Be careful not to disturb the pellets.

 

SECTION: Alkaline Lysis
11. Add 7 mL of ZymoPURE P1 (Red) to each bacterial cell pellet and resuspend completely by pipetting. Combine into one bottle when cells are completely resuspended.

SECTION: Alkaline Lysis
12. Add 14 mL of ZymoPURE P2 (Green) and immediately mix by gently inverting the tube 6 times.

 

SECTION: Alkaline Lysis
13. Add 14 mL of ZymoPURE P3 (Yellow) and mix gently but thoroughly by inversion.

 

SECTION: Alkaline Lysis
14. Ensure the plug is attached to the Luer Lock at the bottom of the ZymoPURE Syringe Filter. Place the syringe filter upright in a tube rack and load the lysate into the ZymoPURE Syringe Filter and wait 8 min for the precipitate to float to the top.

SECTION: Alkaline Lysis
15. Remove the Luer Lock plug from the bottom of the syringe and place it into a clean 50 mL conical tube. Place the plunger in the syringe and push the solution through the ZymoPURE Syringe Filter in one continuous motion until approximately 33 mL-35 mL of cleared lysate is recovered. Save the cleared lysate!

SECTION: Alkaline Lysis
16. Add 14 mL ZymoPURE Binding Buffer to the cleared lysate from step 5 and mix thoroughly by inverting the capped tube 10 times.

SECTION: Alkaline Lysis
17. Ensure the connections of the Zymo-Spin V-P Column Assembly are finger-tight and place onto a vacuum manifold.
SECTION: Alkaline Lysis
18. With the vacuum off, add the entire mixture from step 16 into the Zymo-Spin V-P Column Assembly, and then turn on the vacuum until all the liquid has passed completely through the column.
SECTION: Alkaline Lysis
19. Remove and discard the 50 mL reservoir from the top of the Zymo-Spin V-P Column Assembly.

SECTION: Alkaline Lysis
20. With the vacuum off, add 5 mL of ZymoPURE Wash 1 to the 15 mL Conical Reservoir in the Zymo-Spin V-P Column Assembly. Turn on the vacuum until all the liquid has passed completely through the column.

SECTION: Alkaline Lysis
21. With the vacuum off, add 5 mL of ZymoPURE Wash 2 to the 15 mL Conical Reservoir. Turn on the vacuum until all the liquid has passed completely through the column. Repeat this wash step.

SECTION: Alkaline Lysis
22. Remove and discard the 15 ml Conical Reservoir and place the Zymo-Spin V-P Column in a Collection Tube. Centrifuge at 10000 x g, 1 min , in a microcentrifuge, to remove any residual wash buffer.

SECTION: Alkaline Lysis
23. Pre-warm 450 µL of 10 mM Tris-Cl to 50 °C. Transfer the column into a clean 1.5ml microcentrifuge tube and add the 450 µL of 10 mM Tris-Cl directly to the column matrix. Wait 10 min, and then centrifuge at 10000 x g, 1 min in a microcentrifuge.

SECTION: Alkaline Lysis
24. Add 450 µL of PEG buffer to the tube containing sample. Mix by flicking and rotating the 1.5 ml microcentrifuge tube.

SECTION: Alkaline Lysis
25. Centrifuge at 10000 x g, 30 min, 4 °C, in a microcentrifuge.

SECTION: Alkaline Lysis
26. Remove supernatant from the tube without disturbing the pellet.
SECTION: Alkaline Lysis
27. Add 1 mL of 70% EtOH and spin 10000 x g, 10 min, 4 °C, in a microcentrifuge.

SECTION: Alkaline Lysis
28. Repeat step 26 and 27. 

SECTION: Alkaline Lysis
29. Remove supernatant and any left over 70% EtOH from microcentrifuge tube.
SECTION: Alkaline Lysis
30. Air dry for 10 min or until no visible liquid is left in the tube.

 

SECTION: Alkaline Lysis
31. Dissolve DNA pellet in 18 µL 10 mM Tris-Cl. 

SECTION: Alkaline Lysis
32. Store DNA at 4 °C for several days until pellet completely dissolves into solution.

SECTION: Alkaline Lysis
33. Check DNA concentration and quality with Qubit or NanoDrop. Aim for concentration ≥ 1 µg/µL, an A260/280 ratio ~ 1.8, and an A260/230 ratio between 2.0 and 2.2.

 

SECTION: MinION Library prep
34. Adjust sample concentration from step 33 to 1 µg/µL with 10 mM Tris-Cl.

 

SECTION: MinION Library prep
35. Using a wide bore pipette tip, slowly aspirate 15 µL into a low retention microcentrifuge tube.

 

SECTION: MinION Library prep
36. In a separate microcentrifuge tube, add 0.5 µL FRA to 4.5 µL 10 mM Tris-Cl. Flick the tube to mix well.

 

SECTION: MinION Library prep
37. Add the diluted FRA solution from step 36 into sample tube from step 35.
SECTION: MinION Library prep
38. Gently flick the tubes a few times to mix.

 

SECTION: MinION Library prep
39. Incubate sample on 30 °C heat block for 35 s, then move the tube to 80 °C heat block. Incubate for 2 min at 80 °C.

SECTION: MinION Library prep
40. Remove the tube from heat block and incubate on icefor 1 min, then move the tube off the ice. Equilibrate to 80 Room temperature, about 1 min.

SECTION: MinION Library prep
41. While the sample is equilibrating to 80 Room temperature, add 4.5 µL 10 mM Tris-Cl to 0.5 µLof RAP. Flick to mix well.

 

SECTION: MinION Library prep
42. Add RAP dilution from step 41 into sample tube. Slowly flick the tube a few times to mix. Keep the sample at 80 Room temperature before loading.

 

SECTION: MinION Library loading
43. Add 30 µLof FLT to one tube of FLB, to make flush buffer, according to the Rapid Sequencing Kit instructions. Vortex the solution to mix , then centrifuge briefly.

 

SECTION: MinION Library loading
44. Perform QC on a new MinION flow cell to check available pores and ensure that a sufficient number of pores are present. If there are fewer than 800 available pores, place the flow cell in storage and use a new MinION flow cell.
SECTION: MinION Library loading
45. Use a P1000 pipette to remove about 20 µL-30 µL of storage buffer from priming pore. Load 800 µL flush buffer via the pore slowly. Wait 5 min.

SECTION: MinION Library loading
46. Lift SpotON cover and load 200 µL flush buffer slowly. Try to dispense at a speed where each bead of liquid is siphoned into the SpotON port as soon as it is visible.

SECTION: MinION Library loading
47. Add 34 µL SQB and 15 µL ddH2O to the sample tube from step 42.

 

SECTION: MinION Library loading
48. Flick the tube gently to mix, then centrifuge briefly to collect library to the bottom of the tube.

 

SECTION: MinION Library loading
49. Slowly aspirate 75 µL of library with a wide bore tip. Very slowly, load the library into SpotON pore drop by drop.

 

SECTION: MinION Library loading
50. Close both priming pores and put the SpotON cover back onto the pore.
SECTION: MinION Library loading
51. After loading the library, leave the flow cell on bench for 45 min before starting the run.

 

 

SECTION: Demultiplex Reads
52. Prepare file of draft clone sequences in fasta format:

 

 

SECTION: Demultiplex Reads
53. Prepare file of vector sequence in fasta format:
 

SECTION: Demultiplex Reads
54. Download fastq formatted reads from the device running MinION control software:
 

SECTION: Demultiplex Reads
55. Align nanopore reads to file of draft sequences to assign nanopore reads to clones by best match. Then, for each clone, “clone_name”,  gather a list of matching read names, then use the file of matching read names to gather the matching reads in fastq format:

 

 

SECTION: Identify Full-length Reads
56. 
 

For each clone, align nanopore reads to file of vector sequence:


 

SECTION: Identify Full-length Reads
57. Search for reads that begin and end with high-quality matches to vector sequence on the same strand – these are full-length reads.

 

SECTION: Identify Full-length Reads
58. For each clone, generate a fastq file of full-length reads, as well as a fasta file of the longest full-length read to use as a scaffold for final assembly.

 

 

SECTION: Generate Consensus Sequence
59. Polish the longest read twice, using the other full-length nanopore reads:

 

SECTION: Generate Consensus Sequence
60. Gather up Illumina, nanopore, and (if available) PacBio reads for each clone.

 

 

SECTION: Generate Consensus Sequence
61. Polish the nanopore consensus sequence, using both long and short reads.

 


 

SECTION: Generate Consensus Sequence
62. Repeat Step 61 four more times, for a total of 5 rounds of polishing
SECTION: Generate Consensus Sequence
63. Align reads one last time to generate SAM format alignments suitable for assembly editors.

 

 

SECTION: Generate Consensus Sequence
64. Combine alignments

 

SECTION: Generate Consensus Sequence
65. Generate database for Gap5

 

 

SECTION: Finishing
66. Open the assembly in Gap5:

 

SECTION: Finishing
67. Select ‘Edit Contig’ from the ‘Edit’ menu.
SECTION: Finishing
68. Resolve discrepancies between Illumina reads and full-length nanopore reads (Fig. 2).

 

 

 

SECTION: Finishing
69. Resolve SSRs by realigning reads around the SSR region. Select reads by clicking on their names on the left hand side of the edit window, and choose ‘Realign Selection’ from the ‘Command’ menu.

 

SECTION: Finishing
70. Remove any vector-sequence contamination at the ends of the clone. In the Gap5 edit window, use the ‘Search’ button to search the consensus sequence for the sequences at the cloning site of your vector. Trim away the vector sequence outside of the restriction sites used to generate your clone library (usually EcoRI, BamHI, or MboI).
SECTION: Finishing
71. Annotate any remaining ambiguities in the clone sequence (e.g., unresolved simple sequence repeats, where neither Illumina or nanopore reads are completely accurate) by compiling a feature table, which will be useful when finished clone sequences are submitted to GenBank.