BAsE-Seq: a method for obtaining long viral haplotypes from short sequence reads

We present a method for obtaining long haplotypes, of over 3 kb in length, using a short-read sequencer, Barcode-directed Assembly for Extra-long Sequences (BAsE-Seq). BAsE-Seq relies on transposing a template-specific barcode onto random segments of the template molecule and assembling the barcoded short reads into complete haplotypes. We applied BAsE-Seq on mixed clones of hepatitis B virus and accurately identified haplotypes occurring at frequencies greater than or equal to 0.4%, with >99.9% specificity. Applying BAsE-Seq to a clinical sample, we obtained over 9,000 viral haplotypes, which provided an unprecedented view of hepatitis B virus population structure during chronic infection. BAsE-Seq is readily applicable for monitoring quasispecies evolution in viral diseases. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0517-9) contains supplementary material, which is available to authorized users.


Oligos
All oligos were ordered from Integrated DNA Technologies (IDT) and were HPLCpurified unless otherwise stated.

Measure viral load by qPCR
The concentration of "full-length" genomes in each HBV sample is measured by quantitative real-time PCR, using six 10-fold dilutions of a linearized clone of fulllength HBV as DNA standards. Quantification is achieved by inference from a standard curve generated using the DNA standards.
1. Generate a 10-fold dilution series of six DNA standards from 10 2 to 10 7 copies/µl using a linearized clone of HBV. Each dilution will be included in triplicate on the qPCR plate.
2. Prepare a master mix of the PCR in a 1.5 mL LoBind tube and mix thoroughly by vortexing. Load each well of the 96-well qPCR plate that will be used for quantitation: Step 1: Perform barcode assignment A unique barcode will be assigned to each strand of the HBV genome using a 2-cycle PCR. A restriction site for SbfI will be introduced at the barcode-proximal end of the genome. The reaction is digested with Exonuclease I and purified with AMPure beads to ensure complete removal of barcoding oligos before Step 2.
1. Set up the following reaction in a 0.2 ml PCR tube. Prepare a master mix, if necessary, and add the HBV DNA at the final step to each tube. Keep the master mix and PCR tubes on ice until the denaturation temperature (94ºC) has been reached on the thermocycler. 5:00 at 94ºC 0:45 at 94ºC 4:00 at 60ºC 7:00 at 68ºC 4ºC forever 3. Add 3 µl of Exonuclease I (20U/µl) to the PCR reaction. Vortex thoroughly to mix. Incubate for 1 hour at 37ºC, followed by heat inactivation for 5 minutes at 98ºC. Proceed to the next step immediately.

Purify the
Step 1 product with 53 µl of AMPure XP beads (bead:DNA ratio = 1). Proceed with AMPure purification and elute the DNA in 50 µl of TE.
Step 2: Clonal amplification of barcode-tagged genomes Barcode-tagged genomes from Step 1 will be clonally amplified by performing PCR using universal primers. To maximize reaction yield, clonal amplification will involve two stages: PCR1 with high cycle number (>20) to pre-amplify the barcode-tagged genomes, followed by PCR2 with low cycle number (<10) to obtain the final product at high yield. We have observed that over-amplification in PCR1 or PCR2 will result in chimeric PCR products due to inefficient amplification during the later cycles of PCR. To minimize chimerism, the optimal cycle number will be determined separately by performing real-time PCR. See Appendix B for a more detailed discussion.
1. If an internal standard will be included in the library, Step 1 products from the internal standard and clinical sample will be mixed (at the desired ratio) at this step to obtain a combined sample containing 40,000 genomes.

5.
After obtaining the optimal cycle number for PCR1, perform a duplicate (Rep1 and Rep2) of PCR1 in the standard thermocycler. The product of Rep1 will be used in steps 7-9 for real-time PCR to identify the optimal cycle number for PCR2, and the product of Rep2 will be used in step 10 as template for PCR2. 7. Set up the following reactions in a 96-well qPCR plate using Rep1 as a template. Each reaction will be performed in triplicate on the qPCR plate. Include a negative control (no template). Seal the qPCR plate and perform a brief centrifugation to collect the reagents in the bottom of the wells. 12. Measure the concentration of the sample using the dsDNA Qubit BR assay kit.
13. Load 1 µl of the sample on a DNA 7500 chip on the Bioanalyzer to verify the presence of a ~3.2 kb product.

y cycles
Step 3: Digest with SbfI Clonally amplified genomes from Step 2 will be digested by SbfI to create a 4-bp 3'overhang at the barcode-proximal end of the amplicon. This will protect the barcodeproximal end of the amplicon from exonuclease activity in Step 4.
Important note: We recommend using 6 µg of Step 2 product for SbfI digest at this step to guarantee sufficient yield for the remaining steps of the protocol. However, this may not be possible if the yield for Step 2 is lower than expected. From our experience, we have successfully generated libraries from as little as 2.5 µg of input at this step. The amount of enzyme used in Step 4 will have to be scaled down proportionally.
3. Transfer the sample to a 1.5 ml LoBind tube and add 90 µl of AMPure XP beads (bead:DNA ratio = 1.8). Proceed with AMPure purification and elute the DNA by adding 53 µl of EB.
Step 4: Unidirectional deletion with Exonuclease III A broad range of deletions will be generated from the barcode-distal end of each molecule using Exonuclease III, followed by blunt-ending with S1 Nuclease. The barcode-proximal ends of the molecules are protected from exonuclease activity by the 3'-overhang generated in Step 3.

Important note: If the amount of input for
Step 3 is less than 6 µg, the amount of Exonuclease III and S1 Nuclease used in this step will have to be scaled down proportionally.
2. Set up the following reaction in a 0.2 ml tube: Step 3 product 52 µl Exonuclease III 10x Reaction Buffer 6 µl 58 µl 3. Pre-warm the tube at 30ºC in a thermocycler for 5 minutes.
4. Using a P2 or P10 pipette, add 2 µl of Exonuclease III, start the timer, then mix as rapidly as possible using a P100 or P200 pipette set at '55 µl'. Take care not to introduce bubbles or splash the reaction on the walls of the tube.
5. At the following 5 time points, remove 12 µl of the reaction and add it to the tubes containing 0.5M EDTA. Mixing thoroughly by pipetting, then perform heat inactivation in a thermocycler at 80ºC for 15 minutes. Leave the Exonuclease III reaction in the 30ºC heat block throughout.