Barcoding and Library Preparation

Each sequencing technology has its own mechanism of sequencing. Third generation of sequencing uses pores through which nucleic acid strand is pulled through to read the genetic information. In order to assure that nucleic acids pass the pore at correct speed and orientation, adaptors with motor proteins need to be attached to ends of cDNA molecules. Motor protein then anneals to the pore and pulls the molecule through.

Other the adaptors, barcodes are also attached to the cDNA sample. In our application, two different samples are sequenced simultaneously using one flow cell. In order to distinguish which molecule belongs to what sample, barcodes (short DNA fragments with known sequence) are ligated to the cDNA. Subsequent bioinformatic analysis allows sorting the reads iáccording to the barcodes and assign them into two distinct samples.

Experiment

Initially, cDNA is treated with Ultra II End Prep (NEB), which performs end-repair and tailing. End-prep assures that all fragments end in blunt ends and that there are no overhangs, end-tailing adds non-template dAMP to 3´end, which is complementary with dT on barcodes, which are ligated in the subsequent step using Blunt/TA ligase. After barcode ligation, adaptors are ligated using Quick T4 Ligation Kit. Library is then ready to be loaded into the flow cell after passing through the checkpoint.

Checkpoint testing

At this stage, the limited amount of material limits ways of assuring that the library preparation was successful. Under normal circumstances, it would be possible to check quality of cDNA library with Nanodrop. But due to rather small volume and concentration, it was decided that only quantity will be measured using Qubit ( (as Nanodrop has shown to not being very accurate below concentrations of 30 ng/µl). Table 1 shows the usual yield in various steps.

Step	Amount [ng]
Input mRNA (per sample)	250
Output cDNA (per sample)	550
After End-Prep (per sample)	400
After barcoding (per sample)	350
Pooled (both samples)	700
Library (both samples)	350

Table 1: Approximate yields of material at various steps of library prep. Values show most common yield that were obtained throughout the different library preparations. Measured by Qubit

Troubleshooting: Are adaptors/barcodes attached properly?

During the library prep, usually about 20% of material was lost in the beads purification step. Interestingly, 50% of all material was lost in the final purification of the library. This step should potentially be optimized as this could be one of the reasons for low throughput.

Hypothesis

It was not certain whether the barcodes and adaptors were attached properly. If some of these steps fails, all the subsequent part would most likely fail as well and therefore lead to low sequencing throughput. In order to test if the library preparation has been done correctly, we prepared a library of standard lambda phage DNA (provided with the kit for troubleshooting). Table 2 shows the yields at the various steps. Interestingly, large amount of material is lost at the adaptor ligation step.

Result

Step	Yield [ng]
Input gDNA	870
After End-prep	750
Adaptor ligation	450

Table 1: Yields at the various steps.

Figure 1: Sequencing throughput. In light green, the actively sewuencing pores are show, dark green are currently empty pores waiting for a molecule, blue and other pores are inactive.

Results in figure 1 suggest that there is relatively small amount of DNA (low sequencing throughput), which can be caused by loss of material during the bead purification step.

Figure 2: Quality score of obtained reads.

Graph shown in figure 2 shows that reads are of high quality. In our actual sequencing runs, reads were always of very low quality. This result suggests that low quality / amount of passed reads is most likely due to input material (cDNA library) rather than to the library preparation itself.

Discussion

We have seen that when library from genomic DNA is performed, sequencing is of decent quality. The throughput is also rather low, but quality of reads is high, something that has never been achieved with our library. We can therefore assume that in general, library preparation has one issue which is common across all experiments. The issue is most likely loss of material during bead purification which leads to lower throughout as not all pores are occupied at all times.