# Illumina NovaSeq BCL 分檔

## 2024.04.12 HD

參考資料:

[bcl2fastq and bcl2fastq2 Conversion Software](https://support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software.html)

軟體環境:

在 clinical 帳號中， base環境。

- clinical (`cmuh1202203`)

or

用 zephy 帳號 (建立連結用)

- zephy (`boson520`)

## 下機目錄:

`/DATA5/NGSRAWDATA/NovaSeq/231102_A00486_0155_BHTWF2DSX7`

### 資料內容範例:

(base) [clinical@SR650-4 231102_A00486_0155_BHTWF2DSX7]$ ls
20231102_WGS25.csv  CopyComplete.txt  InterOp  Recipe    RTAComplete.txt  RunParameters.xml     Thumbnail_Images
Config              Data              Logs     RTA3.cfg  RunInfo.xml      SequenceComplete.txt

上機日期: 20231102
上機樣本:

Sample_ID:
C1000_WGS_ST_0734_GEM
C1000_WGS_ST_0735_GEM
C1000_WGS_ST_0736_GEM
C1000_WGS_ST_0743_GEM
C1000_WGS_ST_0744_GEM
C1000_WGS_ST_0745_GEM
C1000_WGS_ST_0746_GEM
C1000_WGS_ST_0747_GEM
C1000_WGS_ST_0749_GEM
C1000_WGS_ST_0750_GEM
C1000_WGS_ST_0751_GEM
C1000_WGS_ST_0752_GEM
C1000_WGS_ST_0753_GEM
C1000_WGS_ST_0757_GEM
C1000_WGS_ST_0758_GEM
C1000_WGS_ST_0759_GEM
C1000_WGS_ST_0760_GEM
C1000_WGS_ST_0761_GEM
C1000_WGS_ST_0762_GEM
C1000_WGS_ST_0763_GEM
C1000_WGS_ST_0766_GEM
C1000_WGS_ST_0767_GEM
C1000_WGS_ST_0768_GEM
C1000_WGS_ST_0769_GEM
C1000_WGS_ST_0774_GEM

## SAV (Sequencing Analysis Viewer) 資料收集

1. InterOP資料夾

AlignmentMetricsOut.bin
BasecallingMetricsOut.bin
CorrectedIntMetricsOut.bin
EmpiricalPhasingMetricsOut.bin
ErrorMetricsOut.bin
ExtendedTileMetricsOut.bin
ExtractionMetricsOut.bin
FWHMGridMetricsOut.bin
ImageMetricsOut.bin
OpticalModelMetricsOut.bin
PFGridMetricsOut.bin
QMetrics2030Out.bin
QMetricsByLaneOut.bin
QMetricsOut.bin
TileMetricsOut.bin

3. RunInfo.xml
4. RunParameters.xml
5. SampleSheet.csv

## 使用 /DATA5/NGSRAWDATA/NovaSeq/Do_SAV_data.sh 收集資料

bash Do_SAV_data.sh 231102 /DATA5/NGSRAWDATA/NovaSeq

## 通知均泰生資

均泰生資: (Winnie 妍均)

寄送SAV 資料

## 更改 SampleSheet(建立鏈結)

扣除 lan 中 Q30 不好的區域
以回傳的SampleSheet 重新上機

使用zephy 帳號:

`sudo ln -s New_sampleSheet.csv .`

更改部分範例:

ExcludeTilesLane3	1157-1178+1257-1278+1357-1378+1457-1478+1557-1578+1657-1678+2157-2178+2257-2278+2357-2378+2457-2478+2557-2578+2657-2678									

## 本機端運行 SAV

下載SAV:
[Sequencing Analysis Viewer (SAV) Software](https://support.illumina.com/sequencing/sequencing_software/sequencing_analysis_viewer_sav/downloads.html)

### 載入資料夾:

bin 檔放置於InterOP


## SAV 結果: 20231102

### Flow cell chart


### Data by cycle
[圖片.png](https://hackmd.io/_uploads/ryaiXS8XT.png)

### Data by lane


### QScore distribution


### QScore heatmap


### %>Q30 結果圖


## 運行bcl2fastq

### bcl2fastq 指令

cd /DATA5/NGSRAWDATA/NovaSeq/231102_A00486_0155_BHTWF2DSX7

nohup bcl2fastq --sample-sheet 20231102_WGS25_exclude.csv  --no-bgzf-compression -r 12 -w 12 -p 48  --output-dir /DATA5/NGSFASTQ/NovaSeq_20231106_BaseCall/ --no-lane-splitting   > /DATA5/NGSFASTQ/NovaSeq_20231106_BaseCall_ST/nohup.out 

### 執行程序

or

bash /DATA5/NGSRAWDATA/NovaSeq/run_bcl2fastq.sh 231102_A00486_0155_BHTWF2DSX7 20231109_WGS25.csv

run_bcl2fastq.sh

in=$1
date=$(basename $in |cut -d'_' -f1)

out='/DATA5/NGSFASTQ/NovaSeq_'${date}'_BaseCall'

csv=$2

cd $in

# cd /DATA5/NGSRAWDATA/NovaSeq/231102_A00486_0155_BHTWF2DSX7

echo "Do BCL to FASTQ: from $in to $out!"


nohup bcl2fastq --sample-sheet $csv  --no-bgzf-compression -r 12 -w 12 -p 48  --output-dir $out --no-lane-splitting   > $out/nohup.out 2>&1 &

### 輸出路徑

fastq output:

`/DATA5/NGSFASTQ/NovaSeq_20231102_BaseCall`

### fastq QC

bash Do_fastqc.sh NovaSeq_20231102_BaseCall

### fastQC 結果

/DATA5/NGSFASTQ/FastQC_new

### bcl2fastq multiqc

bash /DATA5/NGSFASTQ/Do_multiqc.sh NovaSeq_20231102_BaseCall

### multiqc 結果

/DATA5/NGSFASTQ/MultiQC

[multiqc_report.html](https://drive.google.com/file/d/1PrMW3ySrqdYpW8yMy9kZvH4Fo9xrm2YT/view?usp=sharing)

### 在BCL中尋找樣本

bash BCLfindSample.sh $name

### 備份BCL

bash do_tar2other_new.sh 231102_tar 231102_A00486_0155_BHTWF2DSX7

### 合併fastq

bash Do_merge_fastq.sh fastq1.gz fastq2.gz fastq_merge.gz