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Big News! Annoroad announced the cooperation with Oxford Nanopore, to introduce high-throughput sequencing platform PromethION

Recently, Annoroad announced the official cooperation with Oxford Nanopore Technologies (Oxford Nanopore), to introduce the highest-throughput sequencing platform PromethION, which will enhance the capability of Annoroad in providing genome sequencing service globally. Annoroad strives to serve cooperative partners via various sequencing platforms, which has been applied to agricultural, fundamental research, medical and health research areas.

PromethION sequence

PromethION is the latest high-throughput sequencing platform brought by Oxford Nanopore, which supports the direct sequencing of DNA and RNA with features of real time and long reads. PromethION is able to run 48 flow cells at the same time, and each flow cell contains 3000 channels, thus each run allows at most 144,000 channels to conduct sequencing. The total throughput can be Tb level if all flow cells run simultaneously, which meets the demands of super high-throughput and rapid turnaround time.


Until now, Annoroad possesses large-scale R&D and service capacity, with the establishment of leading experimental system and bio-IT cloud system for big data analysis. The comprehensive sequencing platform includes Oxford Nanopore PromethION, PacBio Sequel, Illumina HiSeq X Ten, NovaSeq 6000 and NextSeq 550AR. In July 2018, the southern center of Annoroad went into operation in Yiwu, Zhejiang province. This event will expand the strategic layout of Annoroad in genome sequencing area, and facilitate to build the hardware basis to develop an international advanced genome center. 


About Annoroad

Annoroad Gene Technology was established in 2012, focusing on NGS technology with industrial application on human medical health and life science research. It is a renowned company among China's Genome industry, and recognized as a national level high-tech enterprise.

AnnoGene, as a registered brand under Annoroad, carries out extensive R&D collaborations with higher education institutions and research institutes worldwide, in multi-omics levels such as genomics, transcriptomics, and epigenetics, with featured products in gene expression analysis, single-cell techniques and chromosome Hi-C technology.

With the rapid development in recent years, Annoroad now has opened a branch in Yiwu, Zhejiang province under the great support of local government, remain committed to providing excellent technical solutions for life science research.


Oxford Nanopore Technologies

Oxford Nanopore was founded in 2005 to develop a disruptive, electronic, single-molecule sensing system based on nanopore science. The novel, electronics-based DNA/RNA sequencing technology is being used for a range of biological research applications. These include large scale human genomics, cancer research, microbiology, plant science and environmental research. Oxford Nanopore’s proprietary technology is fully scalable for any requirement. Small formats such as Flongle address the need for on-demand, rapid, smaller tests or experiments, and can be used in labs or outdoors. The pocket-sized MinION is a powerful and portable sequencing device that can deliver high volumes of long read sequence data.  The benchtop GridION X5 can run up to five MinION flow cells at a time, on-demand, for larger genomics projects. The recently launched PromethION is the largest format for nanopore sequencing, designed to offer on-demand use of up to 48 flow cells – each of which can offer more than 100Gb of sequencing data. 

A Breakthrough of gene editing assisted by Annoroad ——the creation of the world’s first pig model for Huntington’s disease

A team of scientists from Jinan University (JNU) has created the world's first pig model with Huntington's disease (HD) by using CRISPR/Cas9 and somatic nuclear transfer technology. This study was published in Cell on March 29th. Annoroad conducted whole genome sequencing of the pig genomic DNA and prepared the DNA library. It is demonstrated for the first time that overt and typical neurodegeneration seen in human brains can be endogenously expressed in large mammals, which offers great hope to investigate the pathogenesis of neurodegenerative diseases and their therapeutics.  


Figure 1. Scientists with the world's first pig models with Huntington's disease

HD together with Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS) are all age-dependent neurodegenerative diseases, which are caused by the accumulation of misfolded proteins in the brain. HD results from the mutation of a single gene HTT, the feature of that makes it an ideal model for the exploration of multiple neurodegenerative diseases. Although mouse models have been established and widely used in the study of HD, there are still considerable differences between human and rodent brains, leading to an absence of overt and striking hallmark of HD. Compared with other small mammals, pigs are genetically, anatomically and physiologically closer to humans.

The research team led by Professor Xiaojiang Li designed two guide RNAs (gRNAs) to target the pig HTT intron after exon 1 to promote DNA breaks and replaces pig HTT exon 1 with the human exon 1 (containing a 150-CAGs repeat) via homologous recombination. The donor vector carries human exon 1 HTT with 150-CAGs repeat flanked by two homologous pig HTT sequences (1Kb for each left and right arm). Afterward, the gRNAs, Cas9 and the donor vector were used to transfect fetal pig fibroblast cells from a female Rongshui pig to precisely express mutant HTT at the endogenous level.


Figure 2. Schematic diagram of the strategy to generate HD pigs via homologous recombination

A total of 2,430 fibroblast cells were screened and 9 were identified as positive cell clones with human HTT exon 1 inserted at the right locus. One of the positive clones was selected for somatic cell nuclear transfer (SCNT) and obtained 2,880 embryos, which were then transferred into 16 surrogate pigs and finally got 7 piglets naturally delivered. Through PCR analysis, 6 of them carried the human HTT exon 1 with expanded CAG repeats. The female founder (F0) pigs were used to hybridize with wild-type (WT) male Bama pigs to generate F1 pigs. After screening, knocked-in (KI) male F1 pigs were mated with WT female Bama pigs to generate F2 pigs. Over two years of breeding, 15 F1 pigs and 10 F2 pigs were yield, and fortunately, they were all carrying mutant HTT.

By delivering the Cas9 nuclease complexed with a synthetic gRNA into a cell, the cell's genome can be cut at the desired location, allowing existing genes to be removed or replaced by new ones. However, the CRISPR/Cas9 mediated gene editing has a risk of off target, which can alter the function of otherwise intact genes. Once it occurs in the KI pigs, it will be hard to distinguish the pathological features are caused by the knocked-in genes or off-target activities. To solve this problem, the research team cooperated with Annoroad, conducted whole-genome sequencing upon genomic DNAs from the cortical tissues of F1 KI pigs. Most likely off-target loci by HTT gRNA1 and gRNA2 was checked, and the analysis shows no off-target mutations in the cortex of F1 KI pig.


Figure 3. off-target assay and whole genome-sequencing

Then the characterization of the HD KI pigs was focused on F0 and F1 KI pigs to investigate their behavioral and pathological changes. HD KI pigs did not show obvious symptoms before the age of 4 months. By continuously monitoring they started showing some symptoms, including less body weight gain, wrinkled and sagging skin, abnormal walking pattern, respiratory difficulty, irregular breathing pattern and even earlier death. The breath difficulty suggests that respiratory failure could contribute to the death of animals and is consistent with the observation that pulmonary dysfunction and aspiration pneumonia/suffocation are the major cause of the death in HD patients.  


Figure 4. Photo of a symptomatic HD KI pig (F0-5) and a WT pig at 5 months old, the foot-printing assay revealing gait abnormalities in the KI pig (F0-5) compared with the WT control

The most important finding in this study is the presence of robust and selective neurodegeneration in the HD KI pig brains, which mimics the severe and preferential neurodegeneration of the medium spiny neurons in HD patients. Thus, the HD KI pigs are ideal models which share the typical behaviour and pathological features of HD patients. This research also paves an avenue for generating animal models to mimic selective neurodegeneration in other critical neurodegenerative diseases, such as AD and PD, and to develop effective therapeutic strategies.

The off-target assay and whole-genome sequencing (WGS) after CRISPR/Cas9 gene editing were conducted by Annoroad. We have launched topspeed WGS service, which only takes 8 working days to get data. Our high-throughput automated platform is applied to the library preparation for individual WGS, population evolution and GWAS, etc.

WGS for off-target loci uses Cas9 and other similar nucleases to break dsDNA and then test off-target loci using high-throughput sequencing method, which can find loci with mutation rates less than 0.1%. Annoroad can conduct comprehensive off-target assay to gene-editing cells or individuals via CRISPR-WGS, which provides a more quick and accurate way to accomplish and evaluate the CRISPR gene-editing technique.



Yan, S., et al., A Huntingtin Knockin Pig Model Recapitulates Features of Selective Neurodegeneration in Huntington's Disease. Cell, 2018.

Annoroad Facilitates the Milestone Study of Single-cellCytomics ——The First Mouse Cell Atlas - Guo Guoji'sTeam, Zhejiang University, Reported by Cell

On February 22 (U. S. local time), Cellthe world's top jonrnal, published the paper of Guo Guoji's Team, School of Medicine, Zhejiang University“Mapping the Mouse Cell Atlas by Microwell-Seq” online. By using the independently developed Microwell-Seq platform, Professor Guo's team conducted single-cell sorting and library preparation on the cell suspensiosolution of more than 50 kinds of mouse organs and tissuesThrough the single-cell transcriptome analysison more than 400,000 cells, this team has mappedthe world's first mammalian cell atlas (Figure 1), which can be described as a milestone study in the field of single-cell cytomics. Annoroad has cooperated with Professor Guo's team to complete the work related to single-cell transcriptome sequencing, laying a solid foundation for further cooperation.

Figure 1 Article workflow

Figure 1 Article workflow

In this study, Guo Guoji' Team developed the Microwell-Seq platform. At first, they captured cells with an agarose microplate. As each microplate contains 100,000 micro holes, it can capture about 5,000 to 10,000 single-cells simultaneously. Next, through the microscopic examination, they removed the excess cells of a single micro hole with a capillary tube to improve the single-cell capture rate. Then, they added the magnetic beads with barcode and combined them to the captured single-cells. After the lysis bufferis added, the cells bound to different magnetic beads were transferred to the tube of 1.5 mL for the subsequent reverse transcription, 3'end amplification, and library preparation sequencing (Figure 2).

Figure 2 Microwell-Seq Platform Technology Flow

Figure 2 Microwell-Seq Platform Technology Flow

The Microwell-Seq platform created by Guo Guoji's Team has achieved the high-throughput cell capture and hybrid library preparation. While improving the accuracy of existing single-cell technology, the experiment time is largely shortened and the construction cost of single-cell library is reduced. At the same time, the mapping of the world's first mammalian cell atlas also provides a good reference for the construction of the next human cell atlas, which will be of guiding significance.


In 2017, the single-cell sequencing technology has been continuously developed. Many researchers have achieved fruitful results by using the single-cell technology. The presentation of various major programs has alsoignited the new era of single-cell sequencing. In the beginning of 2018, our domestic team has made the contribution of milestonestudy to the field of single-cell cytomics. As a leader in the overall solution of single-cell sequencing technology in China, Annoroad Genes Team has rich experience in the cooperation of single-cell sequencing technology with many research institutes, such as Curie Institute of France, Chinese Academy of Sciences Institute of Zoology, Southwest University, and Nanjing Medical University. It has also participated in a number of single-cell research work, and the relevant research achievements have been published in Science, eLife, Immunity, Nature sub-journals and other international high-level journals (Table 1). We have well prepared the single-cell multi-group study solutions, and we are looking forward to exploring the beauty of life with you.


Table 1 List of Annoroad Single-cell Sequencing Articles

Table 1 List of Annoroad Single-cell Sequencing Articles



Han X, Wang R, Zhou Y, Fei L, Sun H, Lai S, Saadatpour A, Zhou Z, Chen H, Ye F, Huang D, Xu Y, Huang W, Jiang M, Jiang X, Mao J, Chen Y, Lu C, Xie J, Fang Q, Wang Y, Yue R, Li T, Huang H, Orkin SH, Yuan GC, Chen M, Guo G. Mapping the Mouse Cell Atlas by Microwell-seq [J]. Cell.2018, 172: 1091–1107.

Everything You Care about Hi-C in 2017 Is Here

At the beginning of 2017, you may still be learning about what is Hi-C, and by the end of the year, the feedback about the articles of genome you may receive is to assemble the chromosome to get genome of chromosome level. Meanwhile, with the pity of failing to construct mapping population, you find out that Hi-C is not to be neglected, don’t you?

When you look back the countless published articles of genome in 2017, you will find out that Hi-C has played an irreplaceable role in the genome assembly - to assist the assembly of genome to get genome of chromosome level. According to current incomplete articles of genome in the following table, Hi-C would eventually become the classic method for genome assembly.

Tab. 1 Articles about Hi-C


If you think that these articles are not enough to show the significance and necessity of Hi-C in genome assembly, I’m going to summarize the questions you concerned about and the articles as follows, to throw out a minnow to catch a whale.

Ø    Reference genome of chromosome level

When you look up the assembly level of genome in NCBI, you may find three different assembly levels: contig, scaffold and chromosome. For different levels, the available analysis based on the genome is also different. It is generally recommended to assemble the genome to get genome of chromosome level, for the reference genome of chromosome level is the basic requirement for numerous analyses.


Fig. 1 Necessity of Reference Genome of Chromosome Level

These are just analyses related to genomics. In fact, analysis related to epigenetics (e.g. whole genome methylation sequencing) also regards the reference genome of chromosome level as an essential condition. Therefore, it is genome of chromosome level that is the ladder for follow-up study and publication.

Ø    Evaluation of assembly results

Generally, the results of assembly can be illustrated by mounting rate (the proportion of the length of mounted version in draft assembly). Currently, all projects of Annoroad have achieved an average mounting rate of over 90%, and some projects even have a mounting rate of over 98%. However, it is relatively shallow to evaluate the effect of assembly simply by comparing the data before and after being mounted. It can be inferred from the already published articles that, more evaluations intend to carry out consistency analysis on the versions before and after being assembled with genetic map. And the fact is that, currently, Annoroad Gene can not only carry out the above analyses, but also adopt the following methods to evaluate the results of assembly:


Fig. 3 Evaluation Methods for the Results of Hi-C Genome Assembly

Different evaluation methods can show the effect of assembly from different aspects. And interaction heat map is the most common and intuitive one.

Ø    Assembly error correction

If assembly can not only realize the scaffolding of chromosome level, but also correct certain errors in draft assembly, it will significantly improve the quality of current genome assembly and reduce errors in assembly. In 2017, Dudchenko O. et al. put forward to add error correction analysis  based on existing assembly policy, and realized assembly after correction of draft genome by 3d-dna. However, the correction function wasn’t applied well due to the limited universality of this software. Currently, Annoroad Gene has optimized the analysis process based on a piece of common software named LACHESIS, and successfully realized the function of correction in Hi-C. Meanwhile, the assembly effect of practical project has greatly improved after error correction.


Fig. 4 Comparison between Heat Maps of Hi-C before and after Error Correction

Hi-C 3D Genome Sequencing Technique


As the leader of 3D genome sequencing technique, Annoroad Gene continues to improve Hi-C sequencing, perfect the content of Hi-C analysis, and devotes itself to providing Hi-C sequencing service with best quality and highest cost performance for vast science researchers. In 2017, Annoroad has upgraded its Hi-C in database construction, sequencing and analysis with continuous innovation. Annoroad has broken through significant technological difficulties and realized sharp decrease of sequencing data size, increase of success rate of database construction and effective utilization of data, comprehensive improvement of the content of multi-group conjoint analysis, and other important technical progresses.

Ø    Advancing Annoroad Hi-C

In 2017, Annoroad not only broke through the technological difficulties in database construction and sequencing, which significantly reduced the data size of sequencing and continually increased the success rate of database construction and effective utilization of data, but also greatly upgraded the process of Hi-C analysis as follows:

1) Optimize the analysis process of LACHESIS to enable this software to apply to the draft assemblies of different assembly levels, and obviously improve the effect of assembly, to yield real chromosome-length scaffold, which is the reference genome of chromosome level. And compare the published articles while displaying the results and add result statistics after assembly to better display the effect;

2) Evaluate the results of multi-level assemblies, regard the evaluation on interaction heat map as the standard analysis, and provide the colinearity analysis, consistency analysis and accuracy analysis based on the practical project as personalized analysis;

3) Add analysis on error correction of assembly to the analysis process of LACHESIS, to provide a strong help to genome assembly.

Of course, we won’t stop here. We will add more practical analyses in the analysis on Hi-C results later, including the location prediction of special structure of chromosome, to better serve people. You may look forward to it.

Annoroad declared to introduce 10 PacBio Sequel Sequencers to continue consolidating the construction of world’s leading genome center

Recently, Annoroad has announced that it will further invest hundreds of millions of RMB in building a multi-technology gene sequencing platform and a data center as well as continue to introduce 10 PacBio Sequel Generation III sequencing platforms so as to consolidate the construction of the world’s leading genome center. This ambition drives Annoroad to further enhance its capabilities of large-scale genomic services worldwide. As a leader in the gene sequencing industry, Annoroad has always been insisting on the mission of organizing and utilizing genomic information and technology to enhance the life value. We, by providing collaborative services with a variety of sequencing platforms, are dedicated to serving our partners and providing core tools for the research & development and the transformation of achievements obtained in many fields of life sciences such as agriculture, basic research, medicine and health.


By means of its unique Single Molecule & Real Time (SMRT) technology, PacBio Sequel can materialize the direct sequencing of individual DNA molecule and is characterized by the technology advantages such as ultra-long read length, zero GC preference and even coverage, and currently, it has been widely applied in the research fields such as genomics, transcriptomics and epigenomics. Annoroad has accumulated extensive project experience in the field of generation III sequencing services. Based on the extensive application prospect of generation-III sequencing technology, Annoroad will further promote its application in the field of gene sequencing and provide the best solution for scientific research.

As an internationally advanced genome center, Annoroad has already possessed large-scale R&D and service capabilities. At present, it has established a leading experimental system and successfully accomplished the automation of sample bank establishment. Initiatives including introducing Illumina NovaSeq and PacBio Sequel to expand the existing sequencing platform will further expand Annoroad’s strategic map in the field of gene sequencing, so as to establish a hardware foundation for building an internationally leading large-scale genome center and additionally, to gain an important competitive edge for the globalization of gene sequencing operations.

In addition to the continuous improvement of its hardware platform, Annoroad focuses on the trend of big data cloudification. In 2016, the company announced a strategic cooperation agreement with Aliyun (Alibaba’s cloud computing platform) to jointly establish a cloud platform of genetic data, i.e. an Internet application platform providing users with genetic data. In 2017, Annoroad declared to establish a genome database unique to Chinese people - Annoroad Typical Chinese Genomes (ATCG Database) in order to deeply explore the unique and reliable variation in the Chinese population and reveal the rule of genetic mutation in the Chinese population. Subsequently, ATCG database was officially put online on the website of SolarGenomics.com. The genetic variation information of the ATCG database will be further enriched by the introduction of PacBio Sequel platform. Through these strategic deployments, Annoroad will further form an important foundation for service users in the production, storage and analysis etc. of genetic data.

The Hi-C of Annoroad Assisted the Publication of Tartary Buckwheat Genome

Shanxi Academy of Agricultural Sciences, South China Agricultural University and the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences published the  research results of tartary buckwheat genome in the Molecular Plant. The title of the article is 《The Tartary Buckwheat Genome Provides Insights into Rutin Biosynthesis and Abiotic Stress Tolerance》.


                Figure 1. The online publication of the tartary buckwheat genome

A high-quality, chromosome-scale Tartary buckwheat genome sequence of 489.3 Mb that is assembled by combining whole genome shotgun sequencing of both Illumina short reads and single-molecule real time long reads, sequence tags of a large DNA insert fosmid, library Hi-C sequencing data and BioNano genome maps. Annoroad Gene Technology involved in the Hi-C study of this article. The reference genome facilitated the identification of many new genes predicted to be involved in rutin biosynthesis and regulation, aluminum stress resistance, and in drought and cold stress responses.


Figure 2.  The Hi-C of Annoroad assisted the assembly of tartary buckwheat genome



The Tartary buckwheat genome provides insights into rutin biosynthesis and abiotic stress tolerance. DOI: http://dx.doi.org/10.1016/j.molp.2017.08.013


Actual Data From Automated Platforms is Coming

Automated platforms can accelerate the cycle of  Human Whole Exome Sequencing, RNA Sequencing and Whole Genome Resequencing. Now we get the actual data for automated platforms. The details are as follows

Human Whole Exome Sequencing

The capture efficiency is more than 70%, the mapping ratio and coverage are above 99%. The data quality is stable at a high level compared with the traditional method.

Table 1. Actual Data of Human WGS 


RNA Sequencing

The data quality still guarantee high standards . The proportion of Clean Reads is over 96%, and the Q30 of the effective data is more than 90%.

Table 2. Actual Data of RNA Sequencing


 Whole Genome Resequencing

We tested four species of soybean, rice, insects and algae. The proportion of Clean Reads is above 96% and Q30 is above 88%.

Table 3. Actual Data of Whole Genome Resequencing



Automated platforms accelerate cycle, 13 days for library construction and sequencing


Within 14 days after verification of sample quality (without data analysis)

Additional 3 days for data analysis


Within 14 days after verification of sample quality (without data analysis)

Additional 3 days for data analysis


Within 13 days after verification of sample quality   (without data analysis)

Additional 3 days for data analysis


Activity Deadline:2017.12.31

Sample Requirements:Nucleic Acid

Agents and Instrument:Agilent Human All Exon V6 Kit for Human Whole Exome

Sequencing, Illumina   HiSeq for sequencing .

Current breakthrough of Annoroad’s application of target region sequencing on exploring the Molecular prognostic stratification of MDS was published on Blood

AbstractXu, Y., Li, Y., Hou, G., Chen, C., & Yu, L. (2016). Applicationof Next Generation Sequencing for Prognostic Stratification in Myelodysplastic Syndromes. Blood, 128(22), 5559. Accessed December 04, 2016.



American Society of Hematology( ASH) is the biggest global association focused on the etiology and treatment of blood disease. On December 3-6, 2016, The 58th annual meeting of American Society of Hematology was held in Santiago. The conference demonstrated updating progress on hematology research. Annnroad and Chinese PLA General Hospital collaborated making a big progress on application of next generation sequencing for prognostic stratification in Myelodysplastic Syndromes. The research caught attention in the conference and was selected into  abstracts of ASH 2016, and was published on Volume 128 of Blood.

1. Introduction of Myelodysplastic Syndromes

Myelodysplastic Syndromes (MDS) are a group of diverse bone marrow disorders in which the bone marrow does not produce enough healthy blood cells. About 50 percent of the patients will develop with Acute Mvelogenous Leukemia (AML), which is more dangerous.

 MDS has a very high heterogeneity. The occurrence and progression of MDA involves many genes. In clinical research, a lot of somatic gene mutations were found in MDS patients. These mutations are potentially valuable for diagnosis and prognostic stratification of MDS. For example, the mutation of DNMT3A influences the poor prognosis of MDS. The mutation of TP53 is occur more often in complex chromosome. Whereas SF3B1 usually appears in RARS, and it’s bounded up with better prognosis. Some genes, include TET2、RUNX1、ASXL1、DNMT3A、EZH2、N-RAS、SF381, are potential for clinical judgment, as recommanded in NCCN clinical guideline. With research progresses, gene mutation test will play an important role in clinical judgment assisting、refined prognosis assessment and precision medicine.


2. Exploration of MDS prognostic stratification by NGS

Collaborating with Chinese PLA General Hospital, Annoroad designed a gene panel to enrich 111 hematologic disease related genes. Target region sequencing was performed on 125 MDS patients' blood. The mutations to biological and clinical phenotypes was further explored:

1)   89% patients carried at least one mutagenic gene;

2)   Mutations of ASXL1( 16.8%), RUNX1( 14.4%)和TET2( 12%) appear in more than 10% patients;

3)  Mutations of GATA1/2, TP53 and DNMT3A are adverse prognostic factors; mutations of RUNX1, KRAS, NRAS, SRSF2 and TET2 are adverse factors of PFS.

4)  Mutations of KRAS、NRAS、GATA1/2 and DNMT3A independently result in poorer prognosis. But mutation of IDH1/2 is a favor one to PFS.

5)  The analyse of HMAs therapy and patients with mythelation-related gene mutations patients showed that clinical effectiveness is: geneWT with non-HMAs> geneWT with HMAs> genemut with HMAs> genemut with non-HMAs

Target region sequencing enriches specific region of genome or genes to sequence. It is cost effective and efficient. Based on target region sequencing, mutation screening of MDS gene is a feasible and reliable prognostic evaluation method. It will help to make progress in personalized treatment decisions for MDS patients.