Illumina 测序文库上机前准备及测序问题咨询
01
如何将dsDNA文库浓度ng/µl转换为nM
片段分析｜Agilent Technologies 2100、Qsep 100......
通过在生物分析仪（如Agilent Technologies 2100、Qsep 100）上运行该文库，确定文库的平均大小。
浓度测定｜Qpcr 、Qubit......
利用荧光定量仪器测定核酸浓度。
浓度换算｜计算公式
02
文库Pooling、稀释、变性
Pooling｜Illumina Pooling小工具
推荐使用Illumina官网的Pooling小工具，连接如下：
https://support.illumina.com/help/pooling-calculator/pooling-calculator.htm
示例：
稀释变性｜Illumina 官方推荐
关于文库的变性稀释可参见Illumina:
Best practices for manually normalizing library concentrations
连接：
https://support.illumina.com.cn/content/illumina-support/apac/zh_CN/bulletins/2017/03/best-practices-for-manually-normalizing-library-concentrations.html
详细不同仪器方法参照各测序仪说明书
链接：
https://sapac.support.illumina.com/?langsel=/my/
03
如何与Illumina获得技术支持
对于实验中出现的问题可通过邮件：techsupport@illumina.com向Illumina技术咨询。根据问题类型，Illumina会索取相关资料用于查明相关原因。下面为每种仪器提供了Illumina支持人员可能要求的不同文件及其位置的列表。
iSeq 100｜
|
Type of Investigation |
Logs or Files Needed |
Location |
|
Clustering failure* |
SAV data |
D:\Illumina\iSeq Runs\[RunFolder]\ |
|
Thumbnails |
D:\Illumina\iSeq Runs\[RunFolder]\Thumbnail_Images |
|
|
Run quality |
SAV data |
D:\Illumina\iSeq Runs\[RunFolder]\ |
|
Pre-run check failure |
Pre-run check logs |
D:\Illumina\iSeq Pre Run Checks\Logs\PreRunLog.[Date/time].log |
|
System check failures |
System check results |
D:\Illumina\iSeq System Checks\SystemCheckReport.[date/time] |
|
Data transfer to network or BaseSpace Sequence Hub |
UCS logs and accessory files |
C:\ProgramData**\Illumina\UniversalCopyService |
|
*Clustering failure is indicated by an error observed at cycle 7 if running iSeq Control Software v1.4 or higher. If running v1.3 or lower, a cluster failure is indicated by a drop in intensity at cycle 9 and 0% PF (clusters passing filter). |
||
|
**The ProgramData folder may be hidden. To un-hide files, select the View tab in the File Explorer window and make sure Hidden Itemsis selected. |
||
MiniSeq｜
|
Type of Investigation |
Logs or Files Needed |
Location |
|
Cycle 1 failure* |
SAV data |
D:\Illumina\MiniSeq Sequencing Temp\[RunFolder] |
|
Focus images |
D:\Illumina\MiniSeq Sequencing Temp\[RunFolder]\Images |
|
|
Run quality |
SAV data |
D:\Illumina\MiniSeq Sequencing Temp\[RunFolder] |
|
Focus images |
D:\Illumina\MiniSeq Sequencing Temp\[RunFolder]\Images |
|
|
Run aborted with error |
SAV data |
D:\Illumina\MiniSeq Sequencing Temp\[RunFolder] |
|
Cycle logs |
D:\Illumina\MiniSeq Sequencing Temp\[RunFolder]\Logs |
|
|
System check failures |
System check results |
D:\Illumina\SystemCheckResults\SystemCheck ResultsDetails.csv File overwrites with each new check and folders for individual tests by date/time |
|
Data transfer to network |
RTA logs |
D:\Illumina\MiniSeq Sequencing Temp\[RunFolder>]\Data\RTA Logs |
|
Data transfer to BaseSpace Sequence Hub |
BaseSpace Broker logs and accessory files |
D:\Illumina\basespace broker |
|
Data transfer to network or BaseSpace Sequence Hub |
UCS logs and accessory files |
C:\ProgramData**\Illumina\Universal CopyService\ |
|
*Cycle 1 failure is indicated by all cameras becoming disabled at cycle 1 due to an inability to detect clusters |
||
|
**The ProgramData folder may be hidden. To un-hide files, select the View tab in the File Explorer window and make sure Hidden Items is selected. |
||
MiSeq｜
|
Type of Investigation |
Logs or Files Needed |
Location |
|
Cycle 1 failure* |
SAV data |
D:\Illumina\MiSeq Temp\[RunFolder] |
|
Run quality |
SAV data |
D:\Illumina\MiSeq Temp\[RunFolder] |
|
Thumbnails |
D:\Illumina\MiSeq Temp\[RunFolder]\Thumbnail_Images\L001\Cx.1 |
|
|
Run aborted with error |
SAV data |
D:\Illumina\MiSeq Temp\[RunFolder] |
|
Cycle logs |
D:\Illumina\MiSeq Temp\[RunFolder]\Logs |
|
|
Thumbnails |
D:\Illumina\MiSeq Temp\[RunFolder]\Thumbnail_Images\L001\Cx.1 |
|
|
System check failures |
System check results |
D:\DiagResults\[date/time] |
|
Data transfer to network |
RTA logs |
D:\Illumina\MiSeq Temp\[RunFolder]\Data\RTA Logs |
|
Data transfer to BaseSpace Sequence Hub |
BaseSpace Broker logs and accessory files |
D:\Illumina\basespace broker |
|
*Cycle 1 failure can be indicated by one of the following errors observed at cycle 1: Best focus not found; Best focus is too near edge of range; No usable signal found, it is possible clustering has failed; Through-focus peak did not exceed SNR threshold; Z Motor attempt to move outside soft limits |
||
NextSeq 500/550｜
|
Type of Investigation |
Logs or Files Needed |
Location |
|
Cycle 1 failure* |
SAV data |
D:\Illumina\NextSeq Control Software Temp\[RunFolder] |
|
Focus images |
D:\Illumina\NextSeq Control Software Temp\[RunFolder]\Images |
|
|
Run quality |
SAV data |
D:\Illumina\NextSeq Control Software Temp\[RunFolder] |
|
Focus images |
D:\Illumina\NextSeq Control Software Temp\[RunFolder]\Images |
|
|
Run aborted with error |
SAV data |
D:\Illumina\NextSeq Control Software Temp\[RunFolder] |
|
Cycle logs |
D:\Illumina\NextSeq Control Software Temp\[RunFolder]\Logs |
|
|
System check failures |
System check results |
D:\Illumina\SystemCheckResults\SystemCheck ResultsDetails.csv |
|
Data transfer to network |
RTA logs |
D:\Illumina\ NextSeq Control Software Temp\[RunFolder]\Data\RTA Logs |
|
Data transfer to BaseSpace Sequence Hub (for NCS v2.2 or earlier |
BaseSpace Broker logs and accessory files |
D:\Illumina\basespace broker |
|
Data transfer to network or BaseSpace Sequence Hub |
UCS logs and accessory files |
C:\ProgramData**\Illumina\UniversalCopyService\ |
|
*Cycle 1 failure is indicated by all cameras becoming disabled at cycle 1 due to an inability to detect clusters |
||
|
**The ProgramData folder may be hidden. To un-hide files, select the View tab in the File Explorer window and make sure Hidden Items is selected. |
||
NextSeq 1000/2000｜
|
Type of Investigation |
Logs or Files Needed |
Location |
|
Run quality |
SAV data |
/usr/local/illumina/runs/[RunFolder] |
|
Run aborted with error |
SAV data |
/usr/local/illumina/runs/[RunFolder] |
|
Cycle logs |
/usr/local/illumina/runs/[RunFolder]/Logs |
|
|
Pre-run check failures |
Pre-run check logs |
/usr/local/illumina/runs/setup/[RunFolder] |
|
Sample sheet related errors |
Orchestrator logs |
/usr/local/ilumina/logs/instrument-orchestrator |
|
Data transfer to network or BaseSpace Sequence Hub |
UCS logs and accessory files |
/usr/local/illumina/logs/universal-copy-service |
HiSeq 1000/1500/2000/2500｜
|
Type of Investigation |
Logs or Files Needed |
Location |
|
Run quality |
SAV data |
D (FCA) or E (FCB):\Illumina\HiSeq Temp\[RunFolder] |
|
Thumbnails |
D (FCA) or E (FCB):\Illumina\HiSeq Temp\[RunFolder]\L00x\Cy.1 |
|
|
Run aborted with error |
SAV data |
D (FCA) or E (FCB):\Illumina\HiSeq Temp\[RunFolder] |
|
Cycle logs |
D (FCA) or E (FCB):\Illumina\HiSeq Temp\[RunFolder]\Logs |
|
|
Certain hardware issues |
Maintenance logs |
D:\Illumina Maintenance LogsTemperature Log Flow Cell #1.csv (Flow cell temperature for A side) Temperature Log Flow Cell #2.csv (Flow cell temperature for B side) Temperature Log Storage.csv (Chiller temperature) Temperature Log Laser 1 - Smd6000 Green 532 APC.csv (Green laser) Temperature Log Laser 2 - Smd6000 Red 660 APC.csv (Red laser) |
|
Data transfer to network |
RTA logs |
D (FCA): or E (FCB):\Illumina\HiSeq Temp\[RunFolder]\Data\RTA Logs |
|
Data transfer to BaseSpace Sequence Hub |
BaseSpace Broker logs and accessory files |
D:\Illumina\basespace broker |
HiSeq 3000/4000/X｜
|
Type of Investigation |
Logs or Files Needed |
Location |
|
Failure to find fiducial |
Auto center images |
O:\Illumina\HiSeqTemp\[RunFolder]\Images |
|
Run quality |
SAV data |
O:\Illumina\HiSeq Temp\[RunFolder] |
|
Thumbnails |
O:\Illumina\HiSeq Temp\[RunFolder]\L00x\Cy.1 |
|
|
Run aborted with error |
SAV data |
O:\Illumina\HiSeq Temp\[RunFolder] |
|
Cycle logs |
O:\Illumina\HiSeq Temp\[RunFolder]\Logs |
|
|
Certain hardware issues |
Certain hardware issues Maintenance logs |
C:\Illumina Maintenance Logs Temperature Log Flow Cell #1.csv (Flow cell temperature for A side) Temperature Log Flow Cell #2.csv (Flow cell temperature for B side) Temperature Log Storage.csv (Chiller temperature) Temperature Log Laser 1 - Smd6000 Green 532 APC.csv (Green laser) Temperature Log Laser 2 - Smd6000 Red 660 APC.csv (Red laser) |
NovaSeq 6000｜
|
Type of Investigation |
Logs or Files Needed |
Location |
|
Failure to find fiducial |
Auto center images |
C:\ProgramData*\Illumina\NovaSeq\NovaSeq Temp\[RunFolder]\Alignment |
|
Run quality |
SAV data |
C:\ProgramData*\Illumina\NovaSeq\NovaSeq Temp\[RunFolder] |
|
Run aborted with error |
SAV data |
C:\ProgramData*\Illumina\NovaSeq\NovaSeq Temp\[RunFolder] |
|
Cycle logs |
C:\ProgramData*\Illumina\NovaSeq\NovaSeq Temp\[RunFolder]\Logs |
|
|
Data transfer to network or BaseSpace Sequence Hub |
UCS logs and accessory files |
C:\ProgramData*\Illumina\UniversalCopyService\ |
|
*The ProgramData folder may be hidden. To un-hide files, select the View tab in the File Explorer window and make sure Hidden items is selected. |
||
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