https://orcid.org/0000-0002-3351-1910
Figures
Abstract
Next Generation Sequencing (NGS) is a powerful tool getting into the field of clinical examination. Its preliminary application in pre-implantation comprehensive chromosomal screening (PCCS) of assisted reproduction (test-tube baby) has shown encouraging outcomes that improves the success rate of in vitro fertilization. However, the conventional NGS library construction is time consuming. In addition with the whole genome amplification (WGA) procedure in prior, makes the single cell NGS assay hardly be accomplished within an adequately short turnover time in supporting fresh embryo implantation. In this work, we established a concise single cell sequencing protocol, ChromInst, in which the single cell WGA and NGS library construction were integrated into a two-step PCR procedure of ~ 2.5hours reaction time. We then validated the feasibility of ChromInst for overnight PCCS assay by examining 14 voluntary donated embryo biopsy samples in a single sequencing run of Miseq with merely 13M reads production. The good compatibility of ChromInst with the restriction of Illumina sequencing technique along with the good library yield uniformity resulted superior data usage efficiency and reads distribution evenness that ensures precisely distinguish of 6 normal embryos from 8 abnormal one with variable chromosomal aneuploidy. The superior succinctness and effectiveness of this protocol permits its utilization in other time limited single cell NGS applications.
Citation: Gao F-F, Chen L, Bo S-P, Yao Y-X, Xu Z-L, Ding Q-Y, et al. (2021) ChromInst: A single cell sequencing technique to accomplish pre-implantation comprehensive chromosomal screening overnight. PLoS ONE 16(5): e0251971. https://doi.org/10.1371/journal.pone.0251971
Editor: Wei Cui, University of Massachusetts Amherst, UNITED STATES
Received: December 15, 2020; Accepted: May 7, 2021; Published: May 20, 2021
Copyright: © 2021 Gao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The data that support the findings of this study have been deposited into CNGB Sequence Archive (CNSA) of China National GeneBank DataBase (CNGBdb) with accession number CNP0001733. Due to the next-generation sequencing data involving Human Genetic Resources (HGR) information, according to the Regulation of the People's Republic of China on the Administration of Human Genetic Resources, we cannot share data as public data directly. If you want to apply for data access to controlled data, please go to CNGBdb Data Access(CDA) (https://db.cngb.org/data_access/) to apply. If you have any questions, please contact CNGBdb@cngb.org.
Funding: This work was supported by the National Key Research and Development Program (grant nos. 2018YFC1003100). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
With wide application of PCR, FISH and DNA chip technology, medical examination has moved into the age of molecular diagnosis in the past decade. Recently, high throughput sequencing methods had shown very much promising adding in this age. The constantly throughput increasing of Next Generation Sequencing (NGS) keeps lowering the unit cost of data generation that strongly promotes the application of NGS from an academic research tool toward a powerful clinical examination method. One of such pioneer applications of NGS in clinical setting is single cell sequencing based pre-implantation comprehensive chromosome screen (PCCS) during in vitro fertilization (IVF) procedure [1–7].
World widely, the human infertility rate had increased up to 10–15% [8–10]. The assisted reproduction, namely test-tube baby technology, is often the final resolution to against infertility in many cases. However, the success rate of conventional IVF procedure is merely 30–35% in average, and could be even lower to 5–8% in females over age 40 [11–16]. Scientific researches has revealed that embryo chromosomal abnormality is one of major causes of IVF failure [17–20]. It’s estimated that chromosomal abnormalities exist in 40–60% embryos obtained by IVF, and this ratio is increasing with the age of the female [21–28]. Under this circumstance, it’s substantially helpful for IVF success that performs preimplantation genetic testing for aneuploidy (PGT-A) in a PCCS manner, of which a few or even a single cell biopsy from in vitro cultured embryos are examined for genome wide chromosomal aneuploidy, then choses the very embryo without chromosomal abnormalities to implant into the mother’s womb [20, 25, 29–31].
A single cell genome counts for a few picogram DNA only. To obtain sufficient initiative materials for NGS library construction, the single cell genome has to be amplified (whole genome amplification, WGA) to millions fold first. The conventional NGS library construction is a time-consuming process including multiple steps of DNA fragmentation, blunt end generation, adapter ligation, PCR amplification etc. The additional WGA process prior to these steps makes single cell sequencing an even more time-consuming process. On the other hand, there is clinical necessity that takes embryo biopsy on the day 3 (or 5) post in vitro fertilization then implant a high-quality embryo into the mother’s womb on day 4 (or 6). This requires the PGT-A procedure to be accomplished overnight. It’s almost impossible to achieve this requirement by the conventional single cell sequencing procedure described above.
A commercial product (Takara) is able to accomplish single cell WGA simultaneously with the sequencing library construction. The integrated WGA-NGS library construction protocol significantly reduced the time consuming of single cell sequencing thus enable the potential of overnight PGT-A. However, in the Takara’s sequencing library, artificial sequences containing only two types of nucleotide, G and T, were added to flank each DNA fragment to be sequenced. In an Illumina sequencing reaction, the sequencing primer has to extend and read through these artificial bases first then reach to target sequence (Fig 1). Determined by Illumina’s sequencing principle, the signal emission from the first a few base positions of a sequencing read is to be used for cluster recognition to initiate a sequencing run, and the four nucleotide types (A, G, C and T) is preferred to be equally distributed in each of these base positions by default [32]. Therefore, if Takara’s WGA-NGS library product is loaded alone on an Illumina sequencer, as lacking of C and A in the first a few sequencing positions, the cluster recognition will not be accomplished properly thus the sequencing run will end up with failure. The way to solve this problem is to add artificial random fragments, i.e. Phix (Illumina, San Diego, USA), into the sequencing library. In this manner, the library is sequenced in combined with the added in Phix fragments which provides randomness of the four nucleotides thus enables proper cluster recognition. The minimal amount of Phix fragments added in should not be less than 10% of total reads number [33], whereas these reads are certainly not to contribute to effective data.
Sequence of pre-amplification primers is gray shaded. The reverse-complimentary sequence of pre-amplification primer is shown in Bold. The sequences of exponential amplification primer 1 is underlined. The reverse-complimentary sequences of exponential amplification primer 2 is dash-underlined. The sequencing primer (Read 1 primer of Illumina) is indicated by the black arrow. The barcode is a sequence of hexamer nucleotides that is unique for each sample in a given sequencing run. The barcode sequencing primer is indicated by the dashed black arrow. The number 1 to 6 indicate the first 6 base position of a sequencing read, of which the signal emission is used for cluster recognition to initiate a sequencing run (N: either base of A, G, C, or T).
An overnight PGT-A assay is typically a sequencing run required by one particular patient with embryo biopsy samples up to 15. It’s estimated that minimally 500–1000 K effective reads would be needed to confidently reporting aneuploidy for any given chromosome in PGT-A (internal data, unpublished). Therefore, a sequencer with moderate throughput, such as Illumina Miseq (maximal 15–25 M reads production depend on sequencing reagent version) is a reasonable choice for this purpose. To ensure each biopsy sample being covered by sufficient effective reads is essential for a successful PGT-A assay. Wasting reads on Phix fragments is unwise, that even could be risky when a low version sequencing reagent is used, especially once the reads generation is far less than the maximal potential in a practical sequencing run.
Taken above together, we are aiming to simplify single cell sequencing procedure in fulfill overnight PGT-A requirement of IVF clinic and to ensure maximal data utilization efficiency of NGS sequencing. We established an integrated single-cell WGA-NGS library construction protocol named ‘ChromInst’. As sufficient base randomness is designed in the first a few positions of every sequencing read, the NGS library constructed in this manner is able to be loaded alone in an Illumina sequencer, i.e. Miseq, without adding in Phix fragments. The feasibility to perform overnight PCCS by ChromInst was then demonstrated with a mimic setup of clinical examination.
Materials and methods
The strategy of single cell WGA in this work was adapted from Multiple Annealing and Looping Based Amplification Cycles (MALBAC) method [34], while the chemical components of cell lysis, pre-amplification, exponential amplification and thermal cycling program remain the same. The pre-amplification primers were modified (Table 1) to be compatible with the adaptor sequences of Illumina sequencing library (Illumina, San Diego, USA, Table 1). Briefly, a single cell was lysed in a 5 μl reaction with 12.5 μg/ml proteinase K, 30mM Tris-HCl (pH7.8), 0.2% Triton X-100, 20 mM KCl and 2 mM EDTA for 15 min. Subsequently, a 30 μl of pre-amplification mixture containing pre-amplification primers and DNA Polymerase was added to the reaction. After the 12 cycles of pre-amplification program (95°C-2min, 95°C-15s, 15°C-50s, 25°C-40s, 35°C-30s, 65°C-40s and 75°C-40s), another 30 μl of exponential amplification mix containing exponential amplification primers was added and subjected for 17 cycles of exponential amplification (94°C-30s, 94°C-20s, 63°C-30s and 72°C-40s). The resulting WGA product with the format of Illumina NGS sequencing library (Fig 1) was then subject to be sequenced on Illumina platform.
The 27 bp constant sequence of original MALBAC pre-amplification primer is underlined. The 13 bp constant sequence of the 3’ end sequence of Illumina exponential amplification upstream primer used to replace the 27 bp constant sequence of original MALBAC are shown in Italic. In searching optimal primer length for effective sequencing library construction, random nucleotides in variable length were inserted into the pre-amplification primer (design 1–5, Table 1). Barcode is a sequence of hexamer nucleotide unique for each sample in a given sequencing run. “N” represents either base A, G, C, or T.
The works in this study were divided into three phases. In the first phase, 50 picograms human genomic DNA were used as biological sample in protocol optimization. Conventional agarose gel (2%) electrophoresis were used to evaluate the efficiency and size range of WGA product (namely, sequencing library).
In the second phase, single cells of in vitro cultured human lymphocytes (GM12878) were picked by mouth pipetting under stereo microscope, then used as biological samples. Illumina NGS sequencer MiSeq was used to evaluate the quality of sequencing library upon the integrated WAG-NGS library construction process. To apply multiple samples in a single sequencing run, unique barcode sequences were incorporated respectively into the primers of exponential amplification (of WGA) for each sample. After the WGA-NGS library construction, the libraries of each sample were mixed at equal volume and then purified with MagBead DNA Purification Kit (CoWin Biotech, Beijing, China) followed the manufacture’s protocol. The DNA concentration of purified library mixture was determined by Qubit®2.0 (Thermo Fisher Scientific, Waltham, MA, USA) followed the manufacture’s protocol. To determine the amount of library to be loaded into the sequencer, the molarity of the library was calculated from the Qubit results (in nanogram per microliter). The library was sequenced alone without adding of artificial random fragments (Phix). The MiSeq Reagent Kit v3 (theoretical Maximal 25 M reads production) was used and SE75 sequencing program was applied at early ending of 55bp following Illumina’s sequencing protocol.
For data analysis, the sequencing raw data (in.bcl format) was demultiplexed and converted to the FASTQ format using a Perl script configure BclToFastq.pl in CASAVA(Consensus Assessment of Sequence and Variation, version 1.8.2) [35] package based on the sample sheet information. Illumina adaptors, low quality bases (bases with quality score less than 20) were removed from the FASTQ file using Trimmomatic [36] (version 0.35). High quality reads were mapped to human reference genome (hg19) [37] using BWA (Burrows-Wheeler Alignment Tool, version 0.7.12-r1039) with default parameters [38]. The mapped reads were sorted and converted to binary format.bam using SamtoBam.jar in Picard (version1.119) package [38].
In the third phase, trophectoderm biopsy samples of in vitro cultured human embryo donated from an anonymous volunteer were examined to validate the application of the protocol established in clinical setting of overnight PCCS. The human embryo biopsies were collected on May 5, 2017 and testing and analysis were carried out from that day to May 6. We do not have access to information that can identify individual participants during or after data collection. This study was approved by the Ethics Committee of Nanjing Jinling Hospital (reference number 2016NZKY-028-02), and written informed consent was obtained prior to embryo analysis. Biopsy sample duplicates from each embryo were examined separately by the protocol established above and original MALBAC WGA followed by routine library construction based NGS descripted previously [7, 34, 39]. To validate whether the library yield uniformity enables sufficient reads coverage among multiple samples over limited total reads production, MiSeq reagent kit v2 with a theoretical maximal output of 15M reads, was used in the test. In addition to the resulting data observed in the phase two for library quality evaluation, the chromosomal copy number variation (CNV) of each embryo biopsy was analyzed by the program descripted in previous publication [35–38, 40]. Briefly, unique mapped reads were extracted from the alignment reads (.bam file) using Samtools (version 1.2.1) [41]. The whole reference genome was divided into a serial of 200Kb or 1M bins. Reads number, GC content were calculated within each bin. GC bias correction was processed for every 1% GC content by local Perl [42] scripts. The R (version 3.0.0) [43] was used to generate the graphs of the GC corrected relative reads number (RRN) of each bin to visualize copy number variations. The CNV identified was subsequently compared between the protocol established in this work and routine MALBAC-NGS based PGT-A.
Results
In order to combine the MALBAC WGA procedure and Illumina sequencing library construction into an integrated process, the 5’ end 27 bp of MALBAC pre-amplification primer sequence (Table 1 underlined sequence) was firstly replaced by the 3’ end sequence of the Illumina adapter. To determine the optimal sequence length should be used for random nucleotide of pre-amplification primer, length serials of 5bp, 6bp, 7bp, 8bp and 9bp (Table 1, design 1, 2, 3, 4 and 5) were tested. The experimental results showed that, while the design 1 produced amplification product in wider size range and the design 4 and 5 generated lower yield, the design 2 and 3 produces shorter amplification products at higher yield (Fig 2). Thus, the design 2 was chosen for the subsequent protocol development.
Fifty picograns human genomic DNA was used as amplification template. The amplification products are visualized on a 2% agarose gel as routine. M: DM2000 DNA Marker; Lane 1–5: the WGA products of design 1–5.
In the phase two of the study, single cells were picked from in vitro cultured human lymphocytes then the WGA protocol optimized above was applied to each of the cell. Fifteen single cells were used in the test. To minimize examination turnover time, we simply pooled the library from each sample at equal volume instead of the conventional pooling strategy that performs qPCR quantification to each library then pools them in equal molar manner. The pooled library was loaded on MiSeq sequencer without adding artificial random fragments (Phix). The sequencing run was successfully completed that 26M reads at Q30>90% were yield with average length of 55bp. The average reads yield of 15 samples was 1.6M, with maximal reads of 2.3M and minim reads of 1.3M (Table 2). The data that support the findings of this study have been deposited into CNGB Sequence Archive (CNSA) [44] of China National GeneBank DataBase (CNGBdb) with accession number CNP0001733. The evenness of sequencing reads distribution over the samples (less than two folds variation) demonstrated the robustness of the protocol on library yield uniformity. With the unique mapping rate more than 80% (Table 2), the library yield uniformity ensures each single cell sample is covering by sufficient data. At this point, a single cell NGS sequencing protocol was established which was potentially able to accomplish overnight PCCS. We called it ‘ChromInst’ to emphasize its capability to perform chromosomal screening in a swift manner.
To validate whether the ChromInst protocol is indeed able to achieve overnight PCCS as clinically needed, 14 embryo biopsy samples donated by an anonymous volunteer were tested in a single MiSeq run. Based on our previous PGT-A application, a minimal read of 500K is adequate for comprehensive chromosomal aneuploidy screening (unpublished data). As the phase two results showed satisfied library uniformity among samples, we decided to perform the phase three test in a low read yield manner by using MiSeq reagent kit v2 that gives maximal reads production at 15M only, in examining the practicability of the protocol.
While the embryo biopsy samples were arrived in our laboratory at 6 pm, the WGA-NGS library construction was accomplished at 9:30 pm. After equal volume pooling, purification and quantification, the library mixture of 14 samples (in addition with positive and negative controls) was loaded onto the MiSeq sequencer at 11:30 pm. The sequencing run was accomplished at 5:30 AM on the next day and data analysis was accomplished at 6:00 AM. The timeline of the whole procedure and major measures to reduce time cost is summarized in the Table 3.
The sequencing run was successfully accomplished with 13M reads at Q30 > 90% and an average read length of 55 bp. The average reads yield per samples was 871K, with maximal reads of 1.42M and minim reads of 541K (Table 4). Chromosomal CNVs were identified in 8 samples of the 14 embryos examined, while the other 6 samples were euploidy genome (Fig 3). No significant differences were observed in the paired comparison between the results above and the routine MALBAC-NGS based assay (data no shown).
Chromosomes are aligned along X axis from 1 to 22 with X and Y at the end. Chromosomal copy number was set as Y axis. Aneuploidy was identified from 8 of the 14 embryos examined, which are as shown in the panel A: Sample 1; B: Sample 4; C: Sample 5; D: Sample 6; E: Sample 7; F: Sample 8; G: Sample 9; H: Sample13. Panel I shows a random example of normal diploid genome (Sample 11).
Discussion
In this work, we established a modified MALBAC protocol namely ChromInst, which combines the single-cell WGA and Illumina NGS library construction into an integrated procedure. In addition to time saving, the good compatibility of ChromInst with Illumina sequencing principle along with its good library yield uniformity provided superior data usage efficiency and reads distribution evenness. All these advantages allowed the feasibility of using ChromInst to perform overnight PCCS on multiple embryo biopsies in a sequencing run with relatively low throughput, such as by MiSeq reagent kit v2.
A collective evidence indicate that PGT-A in PCCS manner is beneficial to improve the success rate of IVF [18, 20, 22, 23, 29, 45–47], and NGS is a promising technique to perform PCCS [1, 2, 5, 6, 48, 49]. Nevertheless, the conventional NGS-PGT-A protocol takes days to accomplish the assay. As the optimal time for embryo biopsy is on the day 5 post in vitro fertilization and the timing for embryo implantation should be on the day 6 [50], in the conventional NGS based PGT-A, the post biopsy embryos have to be frozen in liquid nitrogen before the examination results turnover. The influence of embryo frozen is remaining in controversial [51–55]. A recent study published in the New England Journal of Medicine demonstrated that frozen embryo resulted higher IVF successful rate [53]. However, the same study also revealed that embryo frozen may cause higher chance of delivering overweight babies and three folds higher chance of gestational pre-eclampsia. Therefore, the benefit and risk balance between frozen and fresh embryo implantation is still to be further evaluated. At this moment, both frozen and fresh embryo implantation are applied in clinical practices. Furthermore, for a patient failed of pregnancy in the first attempt of conventional IVF procedure, her leftover embryos (have to be frozen) are worthy to PGT-A prior to the next try of implantation. Once defrosted, one should avoid the embryos from frozen again before implantation when at all possible. In this circumstance, the overnight PGT-A can be the only way to perform PCCS to improve the chance of IVF success in the second attempt of embryo implantation. As the result, it is necessary to establish an overnight PGT-A procedure that enables fresh embryo implantation in the reality.
The WGA amplification of ChromInst is based on that of MALBAC [34]. MALBAC is a relatively new WGA method which was invented in 2012. It’s amplification uniformity and allele dropout (ADO) rate are superior to previous WGA methods, such as Degenerate Oligonucleotide Primer PCR (DOP-PCR) and Multiple Displacement Amplification (MDA) [56–58] for single cell genome amplification. Two major modifications of the ChromInst protocol were substituting the 27 bp constant part of MALBAC’s pre-amplification primer sequence to the 13 bp (Table 1 Design 2) of the 3’ part of Illumina’s library construction adaptor and were extending the straight 5 “N” to 6 “N” (Table 1, Fig 1). There modifications kept the amplicon looping forming feature of MALBAC in the pre-amplification stage thus inherited the innate high uniformity feature in MALBAC WGA, while accomplishes the sequencing library construction simultaneously. As illustrated in the Fig 3, the WGA uniformity of ChromInst was sufficient for effectively detecting chromosomal aneuploidy from embryonic biopsy by NGS.
When multiple samples are simultaneously examined on a sequencer with limited reads yield capacity, the reads number uniformity over each sample in the sequencing run is important to ensure all samples covered by sufficient sequencing data. Normally, the good uniformity of inter-sample reads yield is achieved by qPCR quantification to the sequencing library of each sample, then pool the samples in equal molar manner. The qPCR is however not generally available in the clinical laboratory setting and the extra time cost is highly undesirable for an overnight assay constrained by time. In our protocol presented, by merit of the reaction constancy of ChromInst, sequencing library of each sample was simply pooled at equal volume prior to load into the sequencer. And the resulted reads yield uniformity was fairly acceptable in term of either reads number on each sample or reads number CV among samples (Tables 2 and 4). Consequently, the ploidy of the 14 tested embryos, including 6 euploidy and 8 aneuploidy samples, were all well identified by merely 13M total reads yield from the sequencing run (Fig 3). The concise experimental protocol and satisfactory examination results demonstrated that the presented approaches are well practical to accomplish PCCS overnight in clinical laboratory setting.
In principle, the sequencing library construction of ChromInst belongs to the category of “random short fragmentation” that is uncapable of detecting the chromosomal structural rearrangement, such as inversion and translocation. In Mate-Pair sequencing, self-cyclization of long DNA fragment is conducted prior to library construction, thus chromosomal structural rearrangement can be identified by sophisticated strategy in subsequent sequencing data analysis [59]. However, the integration of single cell WGA with Mate-Pair library construction remains a great challenge. We also observed that the ADO rate of ChromInst was not as good as in the original MALBAC (unpublished data), presumably due to the suboptimal design of the pre-amplification primers in comparison to the original MALBAC. For this reason, ChromInst may not be suitable in detecting SNPs or point mutations like other single-cell NGS applications.
In summary, a modified MALBAC protocol, ChromInst, was established in this work, which combines single cell WGA and Illumina sequencing library construction into an integrated procedure that reduces the time cost of WGA plus sequencing library construction to an approximately 2.5 hours reaction time. By maintaining the merit of high amplification uniformity of original MALBAC WGA and additional conciseness and superior data efficiency, ChromInst fulfills the clinical necessity of NGS based overnight PCCS. These advantages are also permitting the application of ChromInst in other pressing single-cell NGS applications.
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