10x genomics RNAseq数据分析实战

10x数据类型：

10x数据类型

每个样本测出3个fastq，通过I1,R1,R2来区别，
下载安装cellranger，
下载所需要的reference，

（一）跑cellranger count

/home/dushiyi/software/biosoftware/cellranger-2.2.0/cellranger count \
--id=ID24 \
--fastqs=/cygene/data/20180810_10x/10x/   \
--sample=WBJPE18018647-1_HMVMKCCXY_L6_WBJPE18018647_20180729_P,WBJPE18018647-2_HMVMKCCXY_L6_WBJPE18018647_20180729_P,WBJPE18018647-3_HMVMKCCXY_L6_WBJPE18018647_20180729_P,WBJPE18018647-4_HMVMKCCXY_L6_WBJPE18018647_20180729_P,WBJPE18018648-1_HMVMKCCXY_L7_WBJPE18018648_20180729_P,WBJPE18018648-2_HMVMKCCXY_L7_WBJPE18018648_20180729_P,WBJPE18018648-3_HMVMKCCXY_L7_WBJPE18018648_20180729_P,WBJPE18018648-4_HMVMKCCXY_L7_WBJPE18018648_20180729_P  \
--transcriptome=/home/dushiyi/database/refdata-cellranger-GRCh38-1.2.0

8个样本，数据量约132G，耗时38小时，线程20个，内存128G。
最后得到的结果在outs目录下

Outputs:
- Run summary HTML:                      /cygene/data/20180810_10x/work/L006/outs/web_summary.html
- Run summary CSV:                       /cygene/data/20180810_10x/work/L006/outs/metrics_summary.csv
- BAM:                                   /cygene/data/20180810_10x/work/L006/outs/possorted_genome_bam.bam
- BAM index:                             /cygene/data/20180810_10x/work/L006/outs/possorted_genome_bam.bam.bai
- Filtered gene-barcode matrices MEX:    /cygene/data/20180810_10x/work/L006/outs/filtered_gene_bc_matrices
- Filtered gene-barcode matrices HDF5:   /cygene/data/20180810_10x/work/L006/outs/filtered_gene_bc_matrices_h5.h5
- Unfiltered gene-barcode matrices MEX:  /cygene/data/20180810_10x/work/L006/outs/raw_gene_bc_matrices
- Unfiltered gene-barcode matrices HDF5: /cygene/data/20180810_10x/work/L006/outs/raw_gene_bc_matrices_h5.h5
- Secondary analysis output CSV:         /cygene/data/20180810_10x/work/L006/outs/analysis
- Per-molecule read information:         /cygene/data/20180810_10x/work/L006/outs/molecule_info.h5
- Loupe Cell Browser file:               /cygene/data/20180810_10x/work/L006/outs/cloupe.cloupe

2018-08-29 03:45:03 [perform] Serializing pipestance performance data.
Waiting 6 seconds for UI to do final refresh.
Pipestance completed successfully!

（二）用dropEst软件跑10x 数据

1，创建目录及配置文件

mkdir -p 01_dropTag 02_alignment 03_dropEst
sh pipeline.sh \
/home/dushiyi/software/biosoftware/dropEst/build  \ # dropest软件路径
/cygene/work/02.dropEst/10x.test.xml \ # 配置文件
/cygene/work/02.dropEst/star \ # star的索引路径
/home/dushiyi/database/refdata-cellranger-GRCh38-1.2.0/genes/genes.gtf  # gtf文件路径

配置文件xml如下：

<config>
    <!-- droptag -->
    <TagsSearch>
        <protocol>10x</protocol>
        <BarcodesSearch>
            <barcode1_length>8</barcode1_length>
            <barcode2_length>16</barcode2_length>
            <umi_length>10</umi_length>
            <r1_rc_length>0</r1_rc_length>
        </BarcodesSearch>

        <Processing>
            <min_align_length>10</min_align_length>
            <reads_per_out_file>10000000</reads_per_out_file>
            <poly_a_tail>AAAAAAAA</poly_a_tail>
        </Processing>
    </TagsSearch>

    <!-- dropest -->
    <Estimation>
        <Merge>
            <barcodes_file>/home/dushiyi/software/biosoftware/dropEst/data/barcodes/10x_aug_2016_split</barcodes_file>
            <barcodes_type>const</barcodes_type>
            <min_merge_fraction>0.2</min_merge_fraction>
            <max_cb_merge_edit_distance>2</max_cb_merge_edit_distance>
            <max_umi_merge_edit_distance>1</max_umi_merge_edit_distance>
            <min_genes_after_merge>100</min_genes_after_merge>
            <min_genes_before_merge>20</min_genes_before_merge>
        </Merge>

        <PreciseMerge>
            <max_merge_prob>1e-5</max_merge_prob>
            <max_real_merge_prob>1e-7</max_real_merge_prob>
        </PreciseMerge>
    </Estimation>
</config>

这里的pipeline.sh如下：

$ cat pipeline.sh
if [ "$#" -ne 4 ]; then
    echo "usage: $0 dropest_directory config_file star_index_folder gtf_with_genes"
    echo "example: $0 ~/dropEst/build ~/dropEst/configs/indrop_v3.xml ~/star/mm10/index/ ~/star/mm10/genes.gtf"
    exit 1
fi

dropest_dir=$1
config_file=$2
star_index=$3
gtf_file=$4
cd 01_dropTag
$dropest_dir/droptag -c $config_file -r 0 -p 20 -S -s -n sample1 -l sample1 /cygene/work/02.dropEst/data/WBJPE18018647-1_HMVMKCCXY_L6_WBJPE18018647_20180729_P_S1_L006_I1_001.fastq.gz /cygene/work/02.dropEst/data/WBJPE18018647-1_HMVMKCCXY_L6_WBJPE18018647_20180729_P_S1_L006_R1_001.fastq.gz /cygene/work/02.dropEst/data/WBJPE18018647-1_HMVMKCCXY_L6_WBJPE18018647_20180729_P_S1_L006_R2_001.fastq.gz
$dropest_dir/droptag -c $config_file -r 0 -p 20 -S -s -n sample2 -l sample2 /cygene/work/02.dropEst/data/WBJPE18018647-2_HMVMKCCXY_L6_WBJPE18018647_20180729_P_S2_L006_I1_001.fastq.gz /cygene/work/02.dropEst/data/WBJPE18018647-2_HMVMKCCXY_L6_WBJPE18018647_20180729_P_S2_L006_R1_001.fastq.gz /cygene/work/02.dropEst/data/WBJPE18018647-2_HMVMKCCXY_L6_WBJPE18018647_20180729_P_S2_L006_R2_001.fastq.gz
$dropest_dir/droptag -c $config_file -r 0 -p 20 -S -s -n sample3 -l sample3 /cygene/work/02.dropEst/data/WBJPE18018647-3_HMVMKCCXY_L6_WBJPE18018647_20180729_P_S3_L006_I1_001.fastq.gz /cygene/work/02.dropEst/data/WBJPE18018647-3_HMVMKCCXY_L6_WBJPE18018647_20180729_P_S3_L006_R1_001.fastq.gz /cygene/work/02.dropEst/data/WBJPE18018647-3_HMVMKCCXY_L6_WBJPE18018647_20180729_P_S3_L006_R2_001.fastq.gz
$dropest_dir/droptag -c $config_file -r 0 -p 20 -S -s -n sample4 -l sample4 /cygene/work/02.dropEst/data/WBJPE18018647-4_HMVMKCCXY_L6_WBJPE18018647_20180729_P_S4_L006_I1_001.fastq.gz /cygene/work/02.dropEst/data/WBJPE18018647-4_HMVMKCCXY_L6_WBJPE18018647_20180729_P_S4_L006_R1_001.fastq.gz /cygene/work/02.dropEst/data/WBJPE18018647-4_HMVMKCCXY_L6_WBJPE18018647_20180729_P_S4_L006_R2_001.fastq.gz
$dropest_dir/droptag -c $config_file -r 0 -p 20 -S -s -n sample5 -l sample5 /cygene/work/02.dropEst/data/WBJPE18018648-1_HMVMKCCXY_L7_WBJPE18018648_20180729_P_S5_L007_I1_001.fastq.gz /cygene/work/02.dropEst/data/WBJPE18018648-1_HMVMKCCXY_L7_WBJPE18018648_20180729_P_S5_L007_R1_001.fastq.gz /cygene/work/02.dropEst/data/WBJPE18018648-1_HMVMKCCXY_L7_WBJPE18018648_20180729_P_S5_L007_R2_001.fastq.gz
$dropest_dir/droptag -c $config_file -r 0 -p 20 -S -s -n sample6 -l sample6 /cygene/work/02.dropEst/data/WBJPE18018648-2_HMVMKCCXY_L7_WBJPE18018648_20180729_P_S6_L007_I1_001.fastq.gz /cygene/work/02.dropEst/data/WBJPE18018648-2_HMVMKCCXY_L7_WBJPE18018648_20180729_P_S6_L007_R1_001.fastq.gz /cygene/work/02.dropEst/data/WBJPE18018648-2_HMVMKCCXY_L7_WBJPE18018648_20180729_P_S6_L007_R2_001.fastq.gz
$dropest_dir/droptag -c $config_file -r 0 -p 20 -S -s -n sample7 -l sample7 /cygene/work/02.dropEst/data/WBJPE18018648-3_HMVMKCCXY_L7_WBJPE18018648_20180729_P_S7_L007_I1_001.fastq.gz /cygene/work/02.dropEst/data/WBJPE18018648-3_HMVMKCCXY_L7_WBJPE18018648_20180729_P_S7_L007_R1_001.fastq.gz /cygene/work/02.dropEst/data/WBJPE18018648-3_HMVMKCCXY_L7_WBJPE18018648_20180729_P_S7_L007_R2_001.fastq.gz
$dropest_dir/droptag -c $config_file -r 0 -p 20 -S -s -n sample8 -l sample8 /cygene/work/02.dropEst/data/WBJPE18018648-4_HMVMKCCXY_L7_WBJPE18018648_20180729_P_S8_L007_I1_001.fastq.gz /cygene/work/02.dropEst/data/WBJPE18018648-4_HMVMKCCXY_L7_WBJPE18018648_20180729_P_S8_L007_R1_001.fastq.gz /cygene/work/02.dropEst/data/WBJPE18018648-4_HMVMKCCXY_L7_WBJPE18018648_20180729_P_S8_L007_R2_001.fastq.gz

cd ../02_alignment
STAR --runThreadN 20 --genomeDir $star_index --readFilesCommand zcat --outSAMtype BAM Unsorted --readFilesIn /cygene/work/02.dropEst/01_dropTag/sample1.fastq.gz.tagged.fastq.gz,/cygene/work/02.dropEst/01_dropTag/sample2.fastq.gz.tagged.fastq.gz,/cygene/work/02.dropEst/01_dropTag/sample3.fastq.gz.tagged.fastq.gz,/cygene/work/02.dropEst/01_dropTag/sample4.fastq.gz.tagged.fastq.gz,/cygene/work/02.dropEst/01_dropTag/sample5.fastq.gz.tagged.fastq.gz,/cygene/work/02.dropEst/01_dropTag/sample6.fastq.gz.tagged.fastq.gz,/cygene/work/02.dropEst/01_dropTag/sample7.fastq.gz.tagged.fastq.gz,/cygene/work/02.dropEst/01_dropTag/sample8.fastq.gz.tagged.fastq.gz

cd ../03_dropEst
# $dropest_dir/dropest -w -M -u -G 20 -g $gtf_file -c $config_file ../02_alignment/Aligned.out.bam
$dropest_dir/dropest -w -m -r "/cygene/work/02.dropEst/01_dropTag/sample8.params.gz /cygene/work/02.dropEst/01_dropTag/sample7.params.gz /cygene/work/02.dropEst/01_dropTag/sample6.params.gz /cygene/work/02.dropEst/01_dropTag/sample5.params.gz /cygene/work/02.dropEst/01_dropTag/sample4.params.gz /cygene/work/02.dropEst/01_dropTag/sample3.params.gz /cygene/work/02.dropEst/01_dropTag/sample2.params.gz /cygene/work/02.dropEst/01_dropTag/sample1.params.gz"  -g $gtf_file -c $config_file ../02_alignment/Aligned.out.bam

分步去跑第一步，droptag，然后将8个样本的结果合并起来，用于第二步的比对，再跑第三步的dropest。
第三步dropest报错：内存超了128G，因为我服务器的运行内存只有128G，转到天河超算中跑。

（三）用zUMIs软件跑10x数据

（四）比较，同一份数据用不同的软件得出的结果比较