NGS library preparation kits, Library의 구축을 신속하고 고효율로 실시 키트
AFT™ Linked-Reads Library Preparation kits
All steps of NGS library prep, including sample DNA Amplification, Fragmentation, and Tagging,
are integrated into a single isothermal AFT™ step. As little as 6.6 pg of genomic DNA is
sufficient for a perfect whole genome sequencing library.
AFT™ technology tolerates inhibitory impurities from raw samples, enables library preparation
directly from 0.1 μL of whole blood and environmental microbe pellet.
Significantly, group of AFT™ amplicons share a common pair-end read, resulting from
running-off amplification of a common mother amplicon, providing a native barcode to link all
the daughter amplicons. The unique feature of linked reads facilitates the long scaffolding, de
novo assembly, and haploid phasing.
AFT™ Linked-Reads Library Preparation technology combines the Amplification,
Fragmentation, and Tagging of target DNA into one reaction, achieves unprecedented
efficiency in DNA sequencing library preparation. It has three significant features:
1. Low DNA input, enables single cell library preparation;
2. Robust to inhibitory impurities in raw samples, enables direct whole blood and
environmental samples library preparations.
3. More significantly, reads are linked by native barcodes, enables long scaffolding, de novo
assembly, and haploid phasing without compartmentation.
AFT™ Linked-Reads Raw Blood WGS Library Preparation Kit의 예
AFT™ Linked-Reads Raw Blood WGS Library preparation technology utilizes multiple strand-
displacement amplification by the strand-displacing enzymes to seamlessly integrate the
Amplification, Fragmentation, and Tagging of the raw blood cell DNA into one reaction,
achieves ultimate efficiency in animal DNA sequencing Library preparation from one tenth of a
Fig. 1. AFT™ primers are degenerate primers with 5’ tag of a fixed sequence. AFT™ primers
randomly bind to multiple sites of the target DNA. Strand-displacing DNA polymerase initiates
the amplification from AFT™ primers; the front amplicons are displaced by the running-up
amplicons and in turn become template for next iteration of AFT™ amplification. The new
amplicons all run off at the 5’ terminus of thetemplate amplicon and share the same sequence
at 3’. In the pair-end sequencing, the common read serves as a native barcode to link all
these that amplify from the same template amplicon.
As a unique feature, groups of AFT™ reads are linked by one common pair-end read that
arise from the running off at the same terminus of the same template. The linked reads
facilitate the long scaffolding, de novo assembly, and chromosome phasing.
Currently, no bioinformatics software package is provided to utilize the linked-reads feature.
AFT™ Linked-Reads Raw Blood WGS Library Preparation Kit seamlessly integrates the blood
DNA extraction, Amplification, Fragmentation, and Tagging into one reaction, achieves ultimate
efficiency in animal DNA sequencing Library preparation. All it takes is 1 uL of blood. The
simple workflow comprises of 3 functional steps: blood cells lysis, AFT reaction of one hour
at 45° C, and Library PCR of 16 cycles,there is also a SPRI beads-based purification before
and after Library PCR step.
Answered Questions ;
Do I need to purify the blood cell DNA for AFT reaction?
No. The strand displacing amplification of AFT reaction tolerates impurities in whole blood very
well, but not well enough to take even 1uL of the whole blood as direct input, it however
works perfectly with 1uL of 1:10 diluted in water.
At what temperature is the kit shipped?
The kit is shipped on gel ice for within US and dry ice for international delivery.
The kit should be stored at -20 °C on receipt.
Can I use 3rd party or homebrew index primer sets to amplify the library?
Yes. If multiplexing, pick and record the index primers from index primer kits (third-party
vendor, NEB E7600S, etc.). Be careful in selecting third-party index primers; some index
primers are based on Nextera transposase recognition sites, not the standard Illumina Read1
and Read2 primer sequences. The correct index primer structures should be like below:
Also, make sure the primers are at 10 uM concentration.
What’s the minimal length of the target DNA?
1,000 bp. Using DNA ladders that include fragments from 100 bp to 50,000bp, we found only
when > 1,000 did the fragments get represented in the sequencing library generated by AFT™
Library Prep kit.
I didn't see the library smear
The possible reasons could be:
1). The DNA might be degraded and reduced to less than 1 kb; AFT ™ technology prefers to
work on DNA longer than 1 kb. Try to keep blood sample on ice or store at low
temperature until the library preparation.
2). Excessive pipetting and vortexing during the workflow might reduce the target DNA to
shorter than 1 kb, avoid excessive pipetting or vortexing.
3) When recovering the elution from SPRI beads, leave at least 3 uL behind, don’t try to
4). The third-party index primers are not compatible; some index primers are based on
Nextera transposase recognition sites, not the standard Illumina Read1 and Read2
sequences. The correct index primer structures should be like below:
Cluster density was low
White cell counts vary dramatically among individuals, try to run PCR with one or two more
PCR duplication rate is high
The PCR duplication rate from AFT™ kit is normally very low, less than 3%, you may
underestimate the input DNA amount and run PCR with too many cycles, try to use fewer PCR
cycles, as long as the library has a molar concentration of greater than 1 nM for 250-500 bp
range, it will be sufficient for a sequencing.