Small RNA Sequencing. Project Workflow. Service Description. Sequencing Service Specification BGISEQ-500 SERVICE OVERVIEW SAMPLE PREPARATION

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1 BGISEQ-500 SERVICE OVERVIEW Small RNA Sequencing Service Description Small RNAs are a type of non-coding RNA (ncrna) molecules that are less than 200nt in length. They are often involved in gene silencing and posttranscriptional regulation of gene expression. Small RNA sequencing is used to discover novel small RNAs, examine the differential expression of all small RNAs and to characterize variations with single-base resolution. Project Workflow We care for your samples from the start through to the result reporting. Highly experienced laboratory professionals follow strict quality procedures to ensure high quality results. Sequencing Service Specification BGISEQ-500 Small RNA Sequencing Services are performed with the BGISEQ-500 sequencing system, featuring cpas and DNA Nanoballs (DNB ) technology¹ for superior data quality. SAMPLE PREPARATION Sample QC 50bp single-end sequencing reads Standard output 20 Million reads per sample Clean data and bioinformatics analysis are available in standard file formats Standard and customized bioinformatics data analysis are available Cloud-based data storage and delivery system LIBRARY CONSTRUCTION SEQUENCING Library QC Turn Around Time Typical 30 working days from sample QC acceptance to data delivery Sequencing QC Expedited services are available, contact your local BGI specialist for details RAW DATA OUTPUT Data QC BIOINFORMATICS ANALYSIS Delivery QC

2 Data Analysis In addition to clean data output, BGI offers a range of standard and customized bioinformatics pipelines for your small RNA sequencing project. Reports and output data files are delivered in industry standard FASTQ, and Excel file formats with publication-ready tables and figures. STANDARD BIOINFORMATICS ANALYSIS Data filtering Length distribution of small RNAs Analysis of common and specific sequences between two samples Small RNAs distribution across selected genome Identification of rrnas, trnas, snrnas, snornas, etc. Identification of repeat associated small RNAs Identification of small RNA sequences which could align to exon/intron Identification of known mirnas by aligning to designated part of mirbase Analysis of the expression pattern of known mirnas Classification of small RNAs into several categories based on customer preference Prediction of novel mirnas and their secondary structures by Mireap and mirdeep from unannotated small RNAs Family analysis of known mirnas CUSTOMIZED ANALYSIS Further customization of Bioinformatics analysis to suit your unique project is available: Please contact your BGI technical representative

3 Small RNA Sequencing Performance Technical reproducibility² To demonstrate the high technical reproducibility of the BGISEQ-500, six human brain samples, two heart samples and two blood samples were sequenced on the BGISEQ-500. Reproducibility was assessed by using six technical replicates of human brain sample (see Fig. 1). The median correlation between the six replicates was 0.98, and the 25% and 75% quantile were 0.98 and 0.99, respectively Fig. 1 - Correlation matrix of brain (six technical replicates), heart (two technical replicates), and blood (two biological replicates) samples, sequenced by the BGISEQ-500 system Expression distribution of mirnas on BGISEQ-500, HiSeq platform and Microarray² Comparisons of the distribution of the sequencing reads in human blood samples on HiSeq to those on the BGISEQ-500 system showed more even coverage of lower abundant mirna species on BGISEQ-500 system, which facilitates the discovery of new mirnas. The diagram in the figure below demonstrates that 90.8% of all blood sequencing reads from the HiSeq match to one single mirna. 98.6% Of all reads from the HiSeq match to the top 10 mirnas. For the BGISEQ-500, 93.1% of all sequenced reads match to the top 10 mirnas. The remaining 6.9% of reads match to the rest of the mirnas.

4 For the Agilent microarray system, the sum of all expression intensities was assumed to be 100% since microarrays show a saturation effect. One of the top-ten mirnas, mir-451a, which is found in 0.8% of HiSeq reads and in 45.9% of BGISEQ-500 reads is the highest expression in microarrays with 37.2% of all expression counts. % of all reads A (ILLUMINA) B (BGISEQ-500) C (AGILENT) 100,0 100,0 100,0 90,0 90,0 90,0 80,0 80,0 80,0 70,0 70,0 70,0 60,0 60,0 60,0 50,0 50,0 50,0 40,0 40,0 40,0 30,0 30,0 30,0 20,0 20,0 20,0 10,0 10,0 10,0 0,0 Percent Reads Illumina 0,0 Percent Reads BGI 0,0 Percent Agilent Others 1,4 Others 6,9 Others 15,7 hsa-mir-142-5p 0,1 hsa-mir-320a 0,6 hsa-mir-150-5p 1,3 hsa-mir-425-5p 0,1 hsa-mir-185-5p 0,9 hsa-mir ,8 hsa-mir-484 0,1 hsa-mir p 1,0 hsa-mir-25-3p 2,5 hsa-mir-22-3p 0,2 hsa-mir-150-5p 1,0 hsa-mir-223-3p 2,9 hsa-mir-16-5p 0,2 hsa-mir-425-5p 1,1 hsa-mir-16-5p 4,6 hsa-mir-92b-3p 0,3 hsa-mir-484 1,5 hsa-mir-15b-5p 5,4 hsa-mir-25-3p 0,4 hsa-mir-486-5p 7,7 hsa-mir-92a-3p 5,8 hsa-mir-451a 0,8 hsa-mir-92a-3p 13,3 hsa-mir ,9 hsa-mir-92a-3p 5,5 hsa-mir-191-5p 20,0 hsa-mir-486-5p 17,0 hsa-mir-486-5p 90,8 hsa-mir-451a 45,9 hsa-mir-451a 37,2 Fig. 2 - Expression distribution of the 10 mirnas with the highest detection in the blood RNA on the (a) HiSeq system, (b) BGISEQ-500, and (c) microarray system. Excellent agreement of known mirna detection between HiSeq and BGISEQ-500 The agreement of known mirna detected of human sample between two platforms is above 80% as demonstrated by the data below (Internal Data). Fig. 3 - Venn diagram of known human mirna detection between HiSeq and BGISEQ-500 platform HiSeq BGISEQ-500

5 Identified known mirna on BGISEQ-500 platform Known mirnas of different species were identified on both BGISEQ-500 and HiSeq platforms in an internal experiment, demonstrating that a consistently higher number of known mirnas was detected on BGISEQ-500, compared to HiSeq platform. This data suggests higher detection sensitivity of mirna for BGISEQ-500. Repeated Rice Rice Repeated Cabbage Cabbage Repeated Pig Pig Repeated Rat Rat Fig. 4 - Comparison of identified known mirna between BGISEQ-500 and HiSeq platform BGISEQ-500 HiSeq Sample Requirements We can process your sample of human, plant, animal, and microbial samples, with the following general requirements: AMOUNT AND CONCENTRATION MINIMUM VOLUME QUANTITATIVE RESULT Total RNA Samples Total RNA 1µg, Concentration 15 ng/ µl 15 µl RIN 7.5;28S/18S 1.3 References 1. Drmanac R, Sparks AB, Callow MJ, Halpern AL, Burns NL, Kermani BG, Carnevali P, Nazarenko I, Nilsen GB, Yeung G, et al. Science(2010) 327(5961):78 81 doi: /science cpas-based sequencing on the BGISEQ-500 to explore small non-coding RNAs. Tobias F, Stefanie R, Chunyu G, et al. Clinical Epigenetics.2016, 8:123. doi: /s

6 Request Information or Quotation Contact your local BGI account representative for the most affordable rates in the industry, to discuss how we can meet your specific project requirements or for expert advice on experiment design, from sample to bioinformatics. International Head Offces United States One Broadway, 3rd Floor Cambridge, MA 02142, USA Tel: Europe Ole Maaløes Vej 3, DK-2200 Copenhagen N, Denmark Tel: Asia Pacific 16 Dai Fu Street, Tai Po Industrial Estate, New Territories, Hong Kong Tel: For BGI s scientific publications relating to BGISEQ-500 Small RNA Sequencing, sample shipping instructions or sample submission forms, please visit our website. Copyright 2017 BGI. The BGI logo is a trademark of BGI. All rights reserved. All brand and product names are trademarks or registered trademarks of their respective holders. Information, descriptions, and specifications in this publication are subject to change without notice. Published January 2017, version 1.0 Services are for research use only.

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