Table 3.

Samples and read processing statistics.

Row:	Pipeline step:		p2 library	p5 library	p8 library
2	Fixed_Seq_Check:	Sequencing reads	90967875	235347955	132988019
3		Matching Fixed_Seq	85407381	221586324	112960113
4		%Matching Fixed_Seq	93.9	94.2	84.9
5	Barcode filtering:	LC & Adapter Pass	85213377	221124118	112733569
6		%LC & Adapter Pass	99.8	99.8	99.8
7		Unique barcodes	8508280	33612246	22583834
8		Barcode groups	7161395	30135828	20674495
9		Reads (row 5) allocated to barcode groups (row 8)	84883356	220848562	112687674
10	Mapping:	Uniquely mapped (incl. rRNA annotation)	72444996	174241156	72111191
11		Multi-mapped on RNA repeats	546788	1873537	625221
12		Multi-mapped (excl. row 11)	6609030	14837602	5038160
13		Unmapped	5282542	29896267	34913102
14		%Mapped	85.99	79.74	64.55
15	Deduplication:	Reads for further use (row 10 + 11)	72991784	176114693	72736412
16		%PCR deduplication, barcode group size limit (of row 15)	9.92	9.44	9.6
17		%Within annotation (of row 16)	92.89	93.55	94.84
18		%Proxy reads (of row 17)	90.48	93.59	89.27
19		Proxy reads (of row 17)	5888752	13988521	5627689
21	Summary:	Proxy reads (row 19)	5888752	13988521	5627689
22		Barcode groups with proxy reads (row 19)	5047022	12993806	4768049
23		Reads in barcode group 1	4323281	12116171	4059143
24		Reads in barcode group 2	1243424	1550542	1163214
25		Reads in barcode group 3	263778	267969	319842
26		Reads in barcode group 4	49468	45988	72452
27		Reads in barcode group 5	8075	7065	11970
28		Reads in barcode group 6	726	786	1068
29		%Proxy reads co-barcoded	26.6	13.4	14.8

Breakdown of read numbers from replicate libraries, pool 2, pool 5 and pool 8, sequenced at different steps of CloseCall processing. Fixed_Seq: Primer sequences for on-bead barcode amplification. LC: low complexity barcode sequences. Of note, Proximity RNA-seq libraries are sequenced close to saturation (approximately 10% of unique reads remain after deduplication) in order to increase the number of detected co-barcoding events. Up to 25% of proxy reads are co-barcoded.