Kun:LabNotes/SingleCellExpr/2013-1-1
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HL140.2 run data analysis[edit]
Lane 2[edit]
- The sequencing libraries were prepared by Rui on Ambion Human Brain Reference (HBR) RNA diluted to various levels [1].
- The multiplexed library was sequenced in Lane 2 of the HL140 flowcell. The first run of this flowcell failed due to low-quality reads towards the second half of Read 1. This flowcell was repeated in 121226_HL140.2.
- Data analysis is performed on 80+8+8bp reads.
Demultiplexing[edit]
- Rui used a combination of three different barcodes for this batch of libraries. Will first decode using the standard N2 index, then with the 8bp barcodes in the T20 primers (correspond to bases 1-8 in Read 2).
- Run the following command in /home/kunzhang/seqStore/121226_HL140.2: 38.1 million of 40.2 million quality-filtered reads were decoded.
qseq2fastqSplitIndxPE96plexGAIIx.pl /media/FreshReads/121226_HL140_2/Data/Intensities/Bustard1.9.4_01-01-2013_root s_2 lane2_decoded > s2_demultiplexing.log &
- In the /home/kunzhang/RNAseq/Data/121226_HL140.2/RL_HBR directory, create eight folders for the eight indexes (N2_indx01, N2_indx02, ..., N2_indx08). Then run the following commands:
./fastq_decon_T20BC8.pl /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_1_Indx1.txt /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_2_Indx1.txt N2_indx01 > N2_indx01_t20bc8.log & ./fastq_decon_T20BC8.pl /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_1_Indx2.txt /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_2_Indx2.txt N2_indx02 > N2_indx02_t20bc8.log & ./fastq_decon_T20BC8.pl /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_1_Indx3.txt /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_2_Indx3.txt N2_indx03 > N2_indx03_t20bc8.log & ./fastq_decon_T20BC8.pl /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_1_Indx6.txt /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_2_Indx6.txt N2_indx06 > N2_indx06_t20bc8.log & ./fastq_decon_T20BC8.pl /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_1_Indx4.txt /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_2_Indx4.txt N2_indx04 > N2_indx04_t20bc8.log & ./fastq_decon_T20BC8.pl /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_1_Indx5.txt /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_2_Indx5.txt N2_indx05 > N2_indx05_t20bc8.log & ./fastq_decon_T20BC8.pl /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_1_Indx7.txt /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_2_Indx7.txt N2_indx07 > N2_indx07_t20bc8.log & ./fastq_decon_T20BC8.pl /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_1_Indx8.txt /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_2_Indx8.txt N2_indx08 > N2_indx08_t20bc8.log &
The 85%-90% of the reads were successfully decoded. N2_index # total reads # decoded reads % decoded N2_indx01 4191926 3752364 89.5% N2_indx02 3539471 3161288 89.3% N2_indx03 5579440 4951974 88.8% N2_indx04 5649610 5053323 89.4% N2_indx05 4926765 4314315 87.6% N2_indx06 2879929 2416443 83.9% N2_indx07 7703629 6637497 86.2% N2_indx08 3696754 3148274 85.2%