TITLE: Preparation of Linked-Read Sequencing Libraries using Haplotagging beads
AUTHORS: marek.kucka, Yingguang Frank Chan

[DESCRIPTION]
Description:
Haplotagging beads are prepared in form of a 96 well plate where each well contains M280-Streptavidin beads linked with complete and barcoded i5 and i7 Tn5-sequencing adapters. Each well contains 884736 well-specific and barcoded Tn5-adapters assembled with Tn5 transposase. Whole plate then has almost 85 million barcodes (96 x 884736 = 84934656 barcodes), with each bead carrying many copies of a single segmented barcode.

The haplotagging beads are used to prepare short-read linked-read sequencing libraries with both i7 and i5 index being 13bp long. High molecule DNA is tagmented with a single bead and all the reads of that DNA molecule will carry the same barcode combination (one of 85 million).

 
 


[STEPS]
SECTION: Linked-read library preparation from High molecular gDNA
2. Dilute gDNA of each sample to 0.15 ng DNA/ul in 10mM Tris, pH=8
Check with HS Qubit concentration of your diluted gDNA using 10 (1.5ng) or 20 (3ng) ul of diluted 0.15ng/ul gDNA

SECTION: Linked-read library preparation from High molecular gDNA
1. Aliquot 1 U of Haplotagging beads per DNA sample.
1 U of Haplotagging beads corresponds to 1 ul of original M280 Streptavidin beads.
Use max of 0.3 ng gDNA per 1 U of Haplotagging beads.
SECTION: Linked-read library preparation from High molecular gDNA
3. WASH buffer
10 mM Tris, pH=8
30 mM NaCl
0.1% Triton X-100
SECTION: Linked-read library preparation from High molecular gDNA
4. TAGMENTATION: mix in same order:

1 U of Haplotagging beads from one well of Haplotagging bead plate
16 ul WASH buffer
2 ul 0.15ng/ul gDNA
5 ul 5xTagmentation buffer

mix by inverting to re-suspend the gDNA with beads
incubate at 55 °C for 10 min 
SECTION: Linked-read library preparation from High molecular gDNA
5. STOP TAGMENTATION and WASH the beads
add 12 ul of 0.6% SDS in each sample
mix by inverting
Room temperature 2 min 
Pulse spin down and place on magnetic stand.
Remove supernatant.
Add 150 ul WASH buffer to the beads, mix by inverting for 1 min 
Place on magnet and repeat the bead washing step one more time.

SECTION: Linked-read library preparation from High molecular gDNA
7. Exonuclease I treatment of beads to remove un-integrated Tn5-transposomes

On magnet remove WASH buffer from the beads
Add 60 ul 1x Exonuclease I buffer and 2 ul Exonuclease I enzyme
Mix well and incubate:
48 °C 24 min 
Mix beads by inverting 5 time every 5 minutes during the incubation
SECTION: Linked-read library preparation from High molecular gDNA
6. Pool or Subsample all samples' beads into one tube.

Pool all or only proportion of beads from each sample depending on the sequencing coverage needed.
Note: 1 ng of DNA needs 30 Gb of sequencing (or 200 million reads) to achieve high enough linked-read information per DNA molecule.

Example: If we tagmentated 10 DNA samples and we will sequenced with 30Gb of sequencing output, then total input amount of DNA from all these 10 samples has to be 1ng. Each sample thus brings in 0.1 ng DNA.
This protocol did tagmentation using 0.3ng DNA, which is why we only pool 1/3 of the beads from each of the 10 samples into one single pooled sample for further preparation, to achieve 1 ng of gDNA input. Keep the rest of the beads in 4 °C as backup. 
SECTION: Linked-read library preparation from High molecular gDNA
8. WASH the beads twice at RT with WASH buffer to remove Exonuclease I
SECTION: Linked-read library preparation from High molecular gDNA
9. Amplification of the libraries (libraries bound to bead surface)

Prepare 50 ul PCR mix for every 5 U of beads:

25 ul of NEBNext‱ High-Fidelity 2X PCR Master Mix / Q5‱ High-Fidelity 2X Master Mix
22.5 ul H2O
2.5 ul 20 uM P5+P7-primer mix

P7 primer AATGATACGGCGACCACCGAGATCTACAC
P5 primer CAAGCAGAAGACGGCATACGAGAT

72 °C 10 min 
98 °C 20 s 
11 cycles of:
98 °C 15 s 
62 °C 30 s 
72 °C 45 s 
final elongation:
72 °C 2 min  

SECTION: Linked-read library preparation from High molecular gDNA
10. run 5 ul of PCR on 2% agarose gel
SECTION: Linked-read library preparation from High molecular gDNA
11. perform Ampure/Home-made magnetic beads size selection on the leftover library to remove library fragments shorter than 300bp and longer than 800bp